Non-endogenous, constitutively activated human serotonin receptors and small molecule modulators thereof

ABSTRACT

Disclosed herein are non-endogenous, constitutively activated forms of the human 5-HT 2A  and human 5-HT 2C  receptors and uses of such receptors to screen candidate compounds. Further disclosed herein are candidate compounds identified by the screening method which act at the 5HT 2A  receptors. Yet further disclosed is a new class of compounds which act at the 5HT 2A  receptors.

This application is a continuation of U.S. patent application Ser. No.10/176,255, filed Jun. 19, 2002, now U.S. Pat. No. 6,846,919 which is adivisional of U.S. patent application Ser. No. 09/767,013, filed Dec.22, 2000 (now issued as U.S. Pat. No. 6,420,541); which is a divisionalof U.S. patent application Ser. No. 09/292,072, filed on Apr. 14, 1999(now issued as U.S. Pat. No. 6,541,209); which is a continuation-in-partof U.S. application Ser. No. 09/060,188, filed Apr. 14, 1998, which is acontinuation-in-part of U.S. Ser. No. 08/839,449, filed Apr. 14, 1997,now abandoned, and claims priority to U.S. Provisional Application No.60/090,783, filed Jun. 26, 1998, now abandoned, U.S. ProvisionalApplication No. 60/112,909, filed Dec. 18, 1998, now abandoned and U.S.Provisional Application No. 60/123,000, filed Mar. 5, 1999, nowabandoned.

FIELD OF THE INVENTION

The present invention relates to non-endogenous, constitutively activeserotonin receptors and small molecule modulators thereof.

BACKGROUND OF THE INVENTION

I. G Protein-Coupled Receptors

G protein-coupled receptors share a common structural motif. All thesereceptors have seven sequences of between 22 to 24 hydrophobic aminoacids that form seven alpha helices, each of which spans the membrane.The transmembrane helices are joined by strands of amino acids having alarger loop between the fourth and fifth transmembrane helix on theextracellular side of the membrane. Another larger loop, composedprimarily of hydrophilic amino acids, joins transmembrane helices fiveand six on the intracellular side of the membrane. The carboxy terminusof the receptor lies intracellularly with the amino terminus in theextracellular space. It is thought that the loop joining helices fiveand six, as well as, the carboxy terminus, interact with the G protein.Currently, Gq, Gs, Gi and Go are G proteins that have been identified.The general structure of G protein-coupled receptors is shown in FIG. 1.

Under physiological conditions, G protein-coupled receptors exist in thecell membrane in equilibrium between two different states orconformations: an “inactive” state and an “active” state. As shownschematically in FIG. 2, a receptor in an inactive state is unable tolink to the intracellular transduction pathway to produce a biologicalresponse. Changing the receptor conformation to the active state allowslinkage to the transduction pathway and produces a biological response.

A receptor may be stabilized in an active state by an endogenous ligandor an exogenous agonist ligand. Recent discoveries such as, includingbut not exclusively limited to, modifications to the amino acid sequenceof the receptor provide means other than ligands to stabilize the activestate conformation. These means effectively stabilize the receptor in anactive state by simulating the effect of a ligand binding to thereceptor. Stabilization by such ligand-independent means is termed“constitutive receptor activation.”

II. Serotonin Receptors

Receptors for serotonin (5-hydroxytryptamine, 5-HT) are an importantclass of G protein-coupled receptors. Serotonin is thought to play arole in processes related to learning and memory, sleep,thermoregulation, mood, motor activity, pain, sexual and aggressivebehaviors, appetite, neurodegenerative regulation, and biologicalrhythms. Not surprisingly, serotonin is linked to pathophysiologicalconditions such as anxiety, depression, obsessive-compulsive disorders,schizophrenia, suicide, autism, migraine, emesis, alcoholism, andneurodegenerative disorders.

Serotonin receptors are divided into seven subfamilies, referred to as5-HT1 through 5-HT7, inclusive. These subfamilies are further dividedinto subtypes. For example, the 5-HT2 subfamily is divided into threereceptor subtypes: 5-HT2A, 5-HT2B, and 5-HT2C. The human 5-HT2C receptorwas first isolated and cloned in 1987, and the human 5-HT2A receptor wasfirst isolated and cloned in 1990. These two receptors are thought to bethe site of action of hallucinogenic drugs. Additionally, antagonists tothe 5-HT2A and 5-HT2C receptors are believed to be useful in treatingdepression, anxiety, psychosis, and eating disorders.

U.S. Pat. No. 4,985,352 describes the isolation, characterization, andexpression of a functional cDNA clone encoding the entire human 5-HT1Creceptor (now known as the 5-HT2C receptor). U.S. Pat. No. 5,661,012describes the isolation, characterization, and expression of afunctional cDNA clone encoding the entire human 5-HT2A receptor.

Mutations of the endogenous forms of the rat 5-HT2A and rat 5-HT2Creceptors have been reported to lead to constitutive activation of thesereceptors (5-HT2A: Casey, C. et al. (1996) Society for NeuroscienceAbstracts, 22:699.10, hereinafter “Casey”; 5-HT2C: Herrick-Davis, K.,and Teitler, M. (1996) Society for Neuroscience Abstracts, 22:699.18,hereinafter “Herrick-Davis 1”; and Herrick-Davis, K. et al. (1997) J.Neurochemistry 69(3): 1138, hereinafter “Herrick-Davis-2”). Caseydescribes a mutation of the cysteine residue at position 322 of the rat5-HT2A receptor to lysine (C322K), glutamine (C322Q), and arginine(C322R) which reportedly led to constitutive activation. Herrick-Davis 1and Herrick-Davis 2 describe mutations of the serine residue at position312 of the rat 5-HT2C receptor to phenylalanine (S312F) and lysine(S312K), which reportedly led to constitutive activation.

SUMMARY OF THE INVENTION

The present invention relates to non-endogenous, constitutivelyactivated forms of the human 5-HT2A and human 5-HT2C receptors andvarious uses of such receptors. Further disclosed are small moleculemodulators of these receptors. Most preferably, these modulators haveinverse agonist characteristics at the receptor.

More specifically, the present invention discloses nucleic acidmolecules and the proteins for three non-endogenous, constitutivelyactivated human serotonin receptors, referred to herein as, AP-1, AP-3,and AP4. The AP-1 receptor is a constitutively active form of the human5-HT2C receptor created by an S310K point mutation. The AP-3 receptor isa constitutively active form of the human 5-HT2A receptor whereby theintracellular loop 3 (IC3) portion and the cytoplasmic-tail portion ofthe endogenous human 5-HT2A receptor have been replaced with the IC3portion and the cytoplasmic-tail portion of the human 5-HT2C receptor.The AP-4 receptor is a constitutively active form of the human 5-HT2Areceptor whereby (1) the region of the intracellular third loop betweenthe proline of the transmembrane 5 region (TM5) and the proline of TM6of the endogenous human 5-HT2A receptor has been replaced with thecorresponding region of the human 5-HT2C receptor (including a S310Kpoint mutation); and (2) the cytoplasmic-tail portion of the endogenoushuman 5-HT2A receptor has been replaced with the cytoplasmic-tailportion of the endogenous human 5-HT2C receptor.

The invention also provides assays that may be used to directly identifycandidate compounds as agonists, partial agonists or inverse agonists tonon-endogenous, constitutively activated human serotonin receptors; suchcandidate compounds can then be utilized in pharmaceuticalcomposition(s) for treatment of diseases and disorders which are relatedto the human 5-HT2A and/or human 5-HT2C receptors.

These and other aspects of the invention disclosed herein will be setforth in greater detail as the patent disclosure proceeds.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following figures, bold typeface indicates the location of themutation in the non-endogenous, constitutively activated receptorrelative to the corresponding endogenous receptor.

FIG. 1 shows a generalized structure of a G protein-coupled receptorwith the numbers assigned to the transmembrane helices, theintracellular loops, and the extracellular loops.

FIG. 2 schematically shows the active and inactive states for a typicalG protein-coupled receptor and the linkage of the active state to thesecond messenger transduction pathway.

FIG. 3 a provides the nucleic acid sequence of the endogenous human5-HT2A receptor (SEQ.ID.NO: 24).

FIG. 3 b provides the corresponding amino acid sequence of theendogenous human 5-HT2A receptor (SEQ.ID.NO: 25).

FIG. 4 a provides the nucleic acid sequence of the endogenous human5-HT2C receptor (SEQ.ID.NO: 26).

FIG. 4 b provides the corresponding amino acid sequence of theendogenous human 5-HT2C receptor (SEQ.ID.NO: 27).

FIG. 5 a provides the nucleic acid sequence of a constitutively activeform of the human 5-HT2C receptor (“AP-1 cDNA”-SEQ.ID.NO: 28).

FIG. 5 b provides the corresponding amino acid sequence of the AP-1 cDNA(“AP-1”-SEQ.ID.NO: 29).

FIG. 6 a provides the nucleic acid sequence of a constitutively activeform of the human 5-HT2A receptor whereby the IC3 portion and thecytoplasmic-tail portion of the endogenous 5-HT2A receptor have beenreplaced with the IC3 portion and the cytoplasmic-tail portion of thehuman 5-HT2C receptor (“AP-3 cDNA”-SEQ.ID.NO: 30).

FIG. 6 b provides the corresponding amino acid sequence of the AP-3 cDNA(“AP-3” SEQ.ID.NO: 31).

FIG. 6 c provides a schematic representation of AP-3, where thedashed-lines represent the portion obtained from the human 5-HT2Creceptor.

FIG. 7 a provides the nucleic acid sequence of a constitutively activeform of the human 5-HT2A receptor whereby (1) the region of the betweenthe proline of TM5 and the proline of TM6 of the endogenous human 5-HT2Areceptor has been replaced with the corresponding region of the human5-HT2C receptor (including a S310K point mutation); and (2) thecytoplasmic-tail portion of the endogenous 5-HT2A receptor has beenreplaced with the cytoplasmic-tail portion of the endogenous human5-HT2C receptor (“AP4 cDNA”—SEQ.ID.NO:32).

FIG. 7 b provides the corresponding amino acid sequence of the AP4 cDNA(“AP4”—SEQ.ID.NO: 33).

FIG. 7 c provides a schematic representation of the mutated 5-HT2Areceptor of FIG. 7 b where the dashed-lines represent the portionobtained from the human 5-HT2C receptor.

FIG. 8 is a representation of the preferred vector, pCMV, used herein.

FIG. 9 is a diagram illustrating (1) enhanced [³⁵S]GTPγS binding tomembranes prepared from COS cells expressing the endogenous human 5-HT2Creceptor in response to serotonin, and (2) inhibition by mianserin usingwheatgerm agglutinin scintillation proximity beads. The concentration of[³⁵S]GTPyS was held constant at 0.3 nM, and the concentration of GDP washeld at 1 μM. The concentration of the membrane protein was 12.5 μg.

FIG. 10 is a diagram showing serotonin stimulation of [³⁵S]GTPγS bindingto membranes expressing AP-1 receptors in 293T cells and the inhibitionby 30 μM mianserin on Wallac™ scintistrips.

FIGS. 11A-B are diagrams showing the effects of protein concentration on[³⁵S]GTPγS binding in membranes pre-pared from 293T cells transfectedwith the endogenous human 5-HT2C receptors and AP-1 receptors comparedto cells transfected with the control vector (pCMV) alone in the absence(A) and presence (B) of 10 μM serotonin. The radiolableled concentrationof [³⁵S]GTPγS was held constant at 0.3 nM, and the GDP concentration washeld constant at 1 μM. The assay was performed on 96-well format onWallac™ scintistrips.

FIG. 12 provides bar-graph comparisons of inositol tris-phosphate(“IP3”) production between the endogenous human 5HT2A receptor and AP-2,a mutated form of the receptor.

FIG. 13 provides bar-graph comparisons of inositol tris-phosphate(“IP3”) production between the endogenous human 5HT2A receptor and AP4,a mutated form of the receptor.

FIG. 14 provides bar graph comparisons of IP3 production between theendogenous human 5-HT2A receptor and AP-3, a mutated form of thereceptor.

FIG. 15 provides bar-graph comparisons of IP3 production between theendogenous human 5-HT2C receptor and AP-1.

FIGS. 16A-C provides representative autoradiograms showing displacementof I¹²⁵-LSD from brain sections by spiperone and compound 116100.

FIGS. 17A-C show in vivo response of animals to 116102 exposure.

DEFINITIONS

The scientific literature that has evolved around receptors has adopteda number of terms to refer to ligands having various effects onreceptors. For clarity and consistency, the following definitions willbe used throughout this patent document. To the extent that thesedefinitions conflict with other definitions for these terms, thefollowing definitions shall control.

AGONISTS shall mean moieties that activate the intracellular responsewhen they bind to the receptor, or enhance GTP binding to membranes.

AMINO ACID ABBREVIATIONS used herein are set out in Table 1:

TABLE 1 ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASPD CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY GHISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINEMET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR TTRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V

PARTIAL AGONISTS shall mean moieties which activate the intracellularresponse when they bind to the receptor to a lesser degree/extent thando agonists, or enhance GTP binding to membranes to a lesserdegree/extent than do agonists.

ANTAGONIST shall mean moieties that competitively bind to the receptorat the same site as the agonists but which do not activate theintracellular response initiated by the active form of the receptor, andcan thereby inhibit the intracellular responses by agonists or partialagonists. ANTAGONISTS do not diminish the baseline intracellularresponse in the absence of an agonist or partial agonist.

CANDIDATE COMPOUND shall mean a molecule (for example, and notlimitation, a chemical compound) which is amenable to a screeningtechnique.

COMPOUND EFFICACY shall mean a measurement of the ability of a compoundto inhibit or stimulate receptor functionality, as opposed to receptorbinding affinity.

CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subject toconstitutive receptor activation.

CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a receptorin the active state by means other than binding of the receptor with itsendogenous ligand or a chemical equivalent thereof.

CONTACT or CONTACTING shall mean bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

ENDOGENOUS shall mean a material that a mammal naturally produces.ENDOGENOUS in reference to, for example and not limitation, the term“receptor” shall mean that which is naturally produced by a mammal (forexample, and not limitation, a human) or a virus. In contrast, the termNON-ENDOGENOUS in this context shall mean that which is not naturallyproduced by a mammal (for example, and not limitation, a human) or avirus. For example, and not limitation, a receptor which is notconstitutively active in its endogenous form, but when manipulatedbecomes constitutively active, is most preferably referred to herein asa “non-endogenous, constitutively activated receptor.” Both terms can beutilized to describe both “in vivo” and “in vitro” systems. For example,and not a limitation, in a screening approach, the endogenous ornon-endogenous receptor may be in reference to an in vitro screeningsystem. As a further example and not limitation, where the genome of amammal has been manipulated to include a non-endogenous constitutivelyactivated receptor, screening of a candidate compound by means of an invivo system is viable.

INHIBIT or INHIBITING, in relationship to the term “response” shall meanthat a response is decreased or prevented in the presence of a compoundas opposed to in the absence of the compound.

INVERSE AGONISTS shall mean moieties that bind the endogenous form ofthe receptor or to the constitutively activated form of the receptor,and which inhibit the baseline intracellular response initiated by theactive form of the receptor below the normal base level of activitywhich is observed in the absence of agonists or partial agonists, ordecrease GTP binding to membranes. Preferably, the baselineintracellular response is inhibited in the presence of the inverseagonist by at least 30%, more preferably by at least 50%, and mostpreferably by at least 75%, as compared with the baseline response inthe absence of the inverse agonist.

LIGAND shall mean an endogenous, naturally occurring molecule specificfor an endogenous, naturally occurring receptor.

PHARMACEUTICAL COMPOSITION shall mean a composition comprising at leastone active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, and not limitation, a human). Those of ordinary skill in theart will understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome based upon the needs of the artisan.

STIMULATE or STIMULATING, in relationship to the term “response” shallmean that a response is increased in the presence of a compound asopposed to in the absence of the compound.

DETAILED DESCRIPTION

I. Particularly Preferred Mutations

For convenience, the sequence information regarding the non-endogenous,constitutively active human 5-HT2A and 5-HT2C receptors are referred toby identifiers as set forth in Table 2:

TABLE 2 IDENTIFIER RECEPTOR SEQ.ID.NO: FIG. AP-1 cDNA 5-HT2C 28 5a AP-15-HT2C 29 5b AP-3 cDNA 5-HT2A 30 6a AP-3 5-HT2A 31 6b AP-4 cDNA 5-HT2A32 7a AP-4 5-HT2A 33 7bAs will be discussed in greater detail below, a mutation analogous tothat reported by Casey (C322K) was utilized in the human 5-HT2A receptorand is referred to herein as AP-2. However, AP-2 did not lead tosufficient constitutive activation to allow for utilization in screeningtechniques.II. Introduction

While it is sometimes possible to make predictions as to the effect ofnucleic acid manipulation from one species to another, this is notalways the case. The results reported by Casey suggest that a pointmutation in the rat 5-HT2A receptor evidences constitutive activation ofthe mutated receptor. Casey reports that the C322K mutation wasapproximately four fold more active than the native rat 5-HT2A receptor.However, for purposes of a most preferred use, i.e., screening ofcandidate compounds, this corresponding mutation in the human 5-HT2Areceptor had little discernable effect in evidencing constitutiveactivation of the human receptor. This, of course, creates thereasonable conclusion that the information reported in Herrick-Davis 1or Herrick-Davis 2 is of limited predictive value relative to themanipulation of the human 5-HT2C receptor. Consequently, the ability tomake reasonable predictions about the effects of mutations to the rat5-HT receptors vis-a-vis the corresponding human receptors is notpossible. Nonetheless, this unfortunate lack of reasonablepredictability provides the opportunity for others to discover mutationsto the human 5-HT receptors that provide evidence of constitutiveactivation.

Therefore, the present invention is based upon the desire of definingmutated sequences of the human serotonin receptors 5-HT2A and 5-HT2Cwhereby such mutated versions of the expressed receptor areconstitutively active. These constitutively active receptors allow for,inter alia, screening candidate compounds.

What has been discovered and disclosed herein is that substantialactivation of the human 5-HT2A receptor can be obtained by “domainswapping,” i.e., by switching the third intracellular domain of the5-HT2A receptor with the third intracellular domain of the 5-HT2Creceptor. Additionally, swapping the cytoplasmic tail of the tworeceptors further increases the IP3 response. Furthermore, mutation ofthe serine at position 310 to lysine (S310K) of the human 5-HT2Creceptor leads to constitutive activation.

What follows is a most preferred approach to identification of candidatecompounds; those in the art will readily appreciate that the particularorder of screening approaches, and/or whether or not to utilize certainof these approaches, is a matter of choice. Thus, the order presentedbelow, set for presentational efficiency and for indication of the mostpreferred approach utilized in screening candidate compounds, is notintended, nor is to be construed, as a limitation on the disclosure, orany claims to follow.

III. Generic G Protein-Coupled Receptor Screening Assay Techniques

When a G protein receptor becomes constitutively active, it binds to a Gprotein (Gq, Gs, Gi, Go) and stimulates the binding of GTP to the Gprotein. The G protein then acts as a GTPase and slowly hydrolyzes theGTP to GDP, whereby the receptor, under normal conditions, becomesdeactivated. However, constitutively activated receptors continue toexchange GDP to GTP. A non-hydrolyzable analog of GTP, [³⁵S]GTPγS, canbe used to monitor enhanced binding to membranes which expressconstitutively activated receptors. It is reported that [³⁵S]GTPγS canbe used to monitor G protein coupling to membranes in the absence andpresence of ligand. An example of this monitoring, among other exampleswell-known and available to those in the art, was reported by Traynorand Nahorski in 1995. The preferred use of this assay system is forinitial screening of candidate compounds because the system isgenerically applicable to all G protein-coupled receptors regardless ofthe particular G protein that interacts with the intracellular domain ofthe receptor.

IV. Confirmation of G Protein-Coupled Receptor Site Screening AssayTechniques

Once candidate compounds are identified using the “generic” Gprotein-coupled receptor assay (i.e. an assay to select compounds thatare agonists, partial agonists, or inverse agonists), further screeningto confirm that the compounds have interacted at the receptor site ispreferred. For example, a compound identified by the “generic” assay maynot bind to the receptor, but may instead merely “uncouple” the Gprotein from the intracellular domain. Thus, by further screening thosecandidate compounds, which have been identified using a “generic” assayin an agonist and/or antagonist competitive binding assay, furtherrefinement in the selection process is provided.

Lysergic acid diethylamide (LSD) is a well-known agonist of the 5-HT2Aand 5-HT2C receptors, while mesulergine is a well-known antagonist tothe 5-HT2C receptor. Accordingly, in most preferred embodiments, anagonist (LSD) and/or antagonist (mesulergine) competitive bindingassay(s) is used to further screen those compounds selected from the“generic” assay for confirmation of serotonin receptor binding.

V. Specified G Protein Assay Techniques

The art-accepted physiologically mediated pathway for the human 5-HT2Aand 5-HT2C receptors is via Gq. Intracellular accumulation of IP3 can beused to confirm constitutive activation of these types of Gq coupledreceptors (see Herrick-Davis-1). As a result, “IP3 accumulation” assayscan be used to further screen those compounds selected from an agonistand/or antagonist competitive binding assay.

VI. Pharmaceutical Compositions

Candidate compounds selected for further development can be formulatedinto pharmaceutical compositions using techniques well known to those inthe art. Suitable pharmaceutically-acceptable carriers are available tothose in the art; for example, see Remington's Pharmaceutical Sciences,16^(th) Edition, 1980, Mack Publishing Co., (Oslo et al., eds.)

EXAMPLES

The following examples are presented for purposes of elucidation, andnot limitation, of the present invention. While specific nucleic acidand amino acid sequences are disclosed herein, those of ordinary skillin the art are credited with the ability to make minor modifications tothese sequences while achieving the same or substantially similarresults reported below. It is intended that equivalent, non-endogenous,constitutively activated human serotonin receptor sequences includethose having eighty-five percent (85%) homology, more preferably havingninety percent (90%) homology, and most preferably having ninety-fivepercent (95%) homology to the disclosed and claimed sequences all fallwithin the scope of any claims appended hereto.

Example 1 Generation of Non-Endogenous, Constitutively Activated HumanSerotonin Receptors 5-HT2C and 5-HT2A

A. Construction of Constitutively Active 5-HT2C Receptor cDNA

1. Endogenous Human 5-HT2C

The cDNA encoding endogenous human 5-HT2C receptor was obtained fromhuman brain poly-A⁺ RNA by RT-PCR. The 5′ and 3′ primers were derivedfrom the 5′ and 3′ untranslated regions and contained the followingsequences:

5′-GACCTCGAGGTTGCTTAAGACTGAAGCA-3′ (SEQ.ID.NO.:1)

5′-ATTTCTAGACATATGTAGCTTGTACCGT-3′ (SEQ.ID.NO.:2)

PCR was performed using either TaqPlus™ precision polymerase(Stratagene) or rTth™ polymerase (Perkin Elmer) with the buffer systemsprovided by the manufacturers, 0.25 μM of each primer, and 0.2 mM ofeach of the four (4) nucleotides. The cycle condition was 30 cycles of94° C. for 1 minute, 57° C. for 1 minute and 72° C. for 2 minutes. The1.5 kb PCR fragment was digested with Xho I and Xba I and subcloned intothe Sal I-Xba I site of pBluescript.

The derived cDNA clones were fully sequenced and found to correspond topublished sequences.

2. AP-1 cDNA

The cDNA containing a S310K mutation (AP-1 cDNA) in the thirdintracellular loop of the human 5-HT2C receptor was constructed byreplacing the Sty I restriction fragment containing amino acid 310 withsynthetic double stranded oligonucleotides encoding the desiredmutation. The sense strand sequence utilized had the following sequence:

5′-CTAGGGGCACCATGCAGGCTATCAACAATGAAAGAAAAGCTAAGAAAGTC-3′ (SEQ.ID.NO: 3)

and the antisense strand sequence utilized had the following sequence:

5′-CAAGGACTTTCTTAGCTTTTCTTTCATTGTTGATAGCCTGCATGGTGCCC-3′ (SEQ.ID.NO: 4)

B. Construction of Constitutively Active 5-HT2A Receptor cDNA

1. Endogenous Human 5-HT2A

The cDNA encoding endogenous human 5-HT2A receptor was obtained byRT-PCR using human brain poly-A⁺ RNA; a 5′ primer from the 5′untranslated region with a Xho I restriction site:

5′-GACCTCGAGTCCTTCTACACCTCATC-3′ (SEQ.ID.NO.5)

and a 3′ primer from the 3′ untranslated region containing an Xba Isite:

5′-TGCTCTAGATTCCAGATAGGTGAAAA CTTG-3′ (SEQ.ID.NO.6).

PCR was performed using either TaqPlus™ precision polymerase(Stratagene) or rTth™ polymerase (Perkin Elmer) with the buffer systemsprovided by the manufacturers, 0.25 μM of each primer, and 0.2 mM ofeach of the four (4) nucleotides. The cycle condition was 30 cycles of94° C. for 1 minute, 57° C. for 1 minute, and 72° C. for 2 minutes. The1.5 kb PCR fragment was digested with Xba I and subcloned into the EcoRV-Xba I site of pBluescript.

The resulting cDNA clones were fully sequenced and found to encode twoamino acid changes from the published sequences. The first change is aT25N mutation in the N-terminal extracellular domain and the secondchange is an H452Y mutation. These mutations are likely to representsequence polymorphisms rather than PCR errors since the cDNA cloneshaving the same two mutations were derived from two independent PCRprocedures using Taq polymerase from two different commercial sources(TaqPlus™ Stratagene and rTth™ Perkin Elmer).

2. Human 5-HT_(2A) (C322K; AP-2)

The cDNA containing the point mutation C322K in the third intracellularloop was constructed by using the Sph I restriction enzyme site, whichencompasses amino acid 322. For the PCR procedure, a primer containingthe C322K mutation:

5′-CAAAGAAAGTACTGGGCATCGTCTTCTTCCT-3′ (SEQ.ID.NO:7)

was used along with the primer from the 3′ untranslated region set forthabove as SEQ.ID.NO:6. The resulting PCR fragment was then used toreplace the 3′ end of the wild type 5-HT_(2A) cDNA by the T4 polymeraseblunted Sph I site. PCR was performed using pfu polymerase (Stratagene)with the buffer system provided by the manufacturer and 10% DMSO, 0.25mM of each primer, 0.5 mM of each of the 4 nucleotides. The cycleconditions were 25 cycles of 94° C. for 1 minute, 60° C. for 1 minute,and 72° C. for 1 minute.

3. AP-3 cDNA

The human 5-HT_(2A) cDNA with intracellular loop 3 (IC3) or IC3 andcytoplasmic tail replaced by the corresponding human 5-HT_(2C) cDNA wasconstructed using PCR-based mutagenesis.

(a) Replacement of IC3 Loop

The IC3 loop of human 5-HT2A cDNA was first replaced with thecorresponding human 5-HT2C cDNA. Two separate PCR procedures wereperformed to generate the two fragments, Fragment A and Fragment B, thatfuse the 5-HT2C IC3 loop to the transmembrane 6 (TM6) of 5-HT2A. The 237bp PCR fragment, Fragment A, containing 5-HT2C IC3 and the initial 13 bpof 5-HT2A TM6 was amplified by using the following primers:

5′-CCGCTCGAGTACTGCGCCGACAAGCTTTGAT-3′ (SEQ.ID.NO:8)

5′-CGATGCCCAGCACTTTCGAAGCTTTTCTTTCATTGTTG-3′(SEQ.ID.NO:9)

The template used was human 5-HT2C cDNA.

The 529 bp PCR fragment, Fragment B, containing the C-terminal 13 bp ofIC3 from 5-HT2C and the C-terminal of 5-HT2A starting at beginning ofTM6, was amplified by using the following primers:

5′-AAAAGCTTCGAAAGTGCTGGGCATCGTCTTCTTCCT-3′ (SEQ.ID.NO:10)

5′-TGCTCTAGATTCCAGATAGGTGAAAACTTG-3′ (SEQ.ID.NO: 11)

The template used was human 5-HT2A cDNA.

Second round PCR was performed using Fragment A and Fragment B asco-templates with SEQ.ID.NO:8 and SEQ.ID.NO: 11 (it is noted that thesequences for SEQ.ID.NOS.: 6 and 11 are the same) as primers. Theresulting 740 bp PCR fragment, Fragment C, contained the IC3 loop ofhuman 5-HT2C fused to TM6 through the end of the cytoplasmic tail ofhuman 5-HT2A. PCR was performed using pfu™ polymerase (Stratagene) withthe buffer system provided by the manufacturer, and 10% DMSO, 0.25 mM ofeach primer, and 0.5 mM of each of the four (4) nucleotides. The cycleconditions were 25 cycles of 94° C. for 1 minute, 57° C. (1st round PCR)or 60° C. (2nd round PCR) for 1 minute, and 72° C. for 1 minute (1stround PCR) or 90 seconds (2nd round PCR).

To generate a PCR fragment containing a fusion junction between thehuman 5-HT2A TM5 and the IC3 loop of 5-HT2C, four (4) primers were used.The two external primers, derived from human 5-HT2A, had the followingsequences:

5′-CGTGTCTCTCCTTACTTCA-3′ (SEQ.ID.NO:12)

The other primer used was SEQ.ID.NO.:6 (see note above regardingSEQ.ID.NOS. 6 and 11). The first internal primer utilized was anantisense strand containing the initial 13 bp of IC3 of 5-HT2C followedby the terminal 23 bp derived from TM5 of 5-HT2A:

5′-TCGGCGCAGTACTTTGATAGTTAGAAAGTAGGTGAT-3′ (SEQ.ID.NO:13)

The second internal primer was a sense strand containing the terminal 14bp derived from TM5 of 5-HT2A followed by the initial 24 bp derived fromIC3 of 5-HT2C:

5′-TTCTAACTATCAAAGTACTGCGCCGACAAGCTTTGATG-3′ (SEQ.ID.NO:14).

PCR was performed using endogenous human 5-HT2A and a co-template,Fragment C, in a 50 ml reaction volume containing 1×pfu buffer, 10%DMSO, 0.5 mM of each of the four (4) nucleotides, 0.25 mM of eachexternal primer (SEQ.ID.NOS. 11 and 12), 0.06 mM of each internal primer(SEQ.ID.NOS. 13 and 14) and 1.9 units of pfu polymerase (Stratagene).The cycle conditions were 25 cycles of 94° C. for 1 minute, 52° C. for 1minute, and 72° C. for 2 minutes and 10 seconds. The 1.3 kb PCR productwas then gel purified and digested with Pst I and Eco RI. The resulting1 kb PstI-Eco RI fragment was used to replace the corresponding fragmentin the endogenous human 5-HT2A sequence to generate the mutant 5-HT2Asequence encoding the IC3 loop of 5-HT2C.

(b) Replacement of the Cytoplasmic Tail

To replace the cytoplasmic tail of 5-HT2A with that of 5-HT2C, PCR wasperformed using a sense primer containing the C-terminal 22 bp of TM7 ofendogenous human 5-HT2A followed by the initial 21 bp of the cytoplasmictail of endogenous human 5-HT2C:

5′-TTCAGCAGTCAACCCACTAGTCTATACTCTGTTCAACAAAATT-3′ (SEQ.ID.NO:15)

The antisense primer was derived from the 3′ untranslated region ofendogenous human 5-HT2C:

5′-ATTTCTAGACATATGTAGCTTGTACCGT-3′ (SEQ.ID.NO:16)

The resulting PCR fragment, Fragment D, contained the last 22 bp ofendogenous human 5-HT2A TM7 fused to the cytoplasmic tail of endogenoushuman 5-HT2C. Second round PCR was performed using Fragment D and theco-template was endogenous human 5-HT2A that was previously digestedwith Acc I to avoid undesired amplification. The antisense primer usedwas SEQ.ID.NO:16 (the sequences for SEQ.ID.NOS. 16 and 2 are the same)and the sense primer used was derived from endogenous human 5-HT2A:

5′-ATCACCTACTTTCTAACTA-3′ (SEQ.ID.NO:17).

PCR conditions were as set forth in Example 1B3(a) for the first roundPCR, except that the annealing temperature was 48° C. and the extensiontime was 90 seconds. The resulting 710 bp PCR product was digested withApa I and Xba I and used to replace the corresponding Apa I-Xba Ifragment of either (a) endogenous human 5-HT2A, or (b) 5-HT2A with 2CIC3 to generate (a) endogenous human 5-HT2A with endogenous human 5-HT2Ccytoplasmic tail and (b) AP-3, respectively.

4. AP4 cDNA

This mutant was created by replacement of the region of endogenous human5-HT2A from amino acid 247, the middle of TM5 right after Pro²⁴⁶, toamino acid 337, the middle of TM6 just before Pro³³⁸, by thecorresponding region of AP-1 cDNA. For convenience, the junction in TM5is referred to as the “2A-2C junction,” and the junction in TM6 isreferred to as the “2C-2A junction.”

Three PCR fragments containing the desired hybrid junctions weregenerated. The 5′ fragment of 561 bp containing the 2A-2C junction inTM5 was generated by PCR using endogenous human 5-HT2A as template,SEQ.ID.NO:12 as the sense primer, and the antisense primer was derivedfrom 13 bp of 5-HT2C followed by 20 bp of 5-HT2A sequence:

5′-CCATAATCGTCAGGGGAATGAAAAATGACACAA-3′ (SEQ.ID.NO:18)

The middle fragment of the 323 bp contains endogenous human 5-HT2Csequence derived from the middle of TM5 to the middle of TM6, flanked by13 bp of 5-HT2A sequences from the 2A-2C junction and the 2C-2Ajunction. This middle fragment was generated by using AP-1 cDNA as atemplate, a sense primer containing 13 bp of 5-HT2A followed by 20 bp of5-HT2C sequences across the 2A-2C junction and having the sequence:

5′-ATTTTTCATTCCCCTGACGATTATGGTGATTAC-3′(SEQ.ID.NO:19);

and an antisense primer containing 13 bp of 5-HT2A followed by 20 bp of5-HT2C sequences across the 2C-2A junction and having the sequence:

5′-TGATGAAGAAAGGGCACCACATGATCAGAAACA-3′ (SEQ.ID.NO:20).

The 3′ fragment of 487 bp containing the 2C-2A junction was generated byPCR using endogenous human 5-HT2A as a template and a sense primerhaving the following sequence from the 2C-2A junction:

5′-GATCATGTGGTGCCCTTTCTTCATCACAAACAT-3′ (SEQ.ID.NO:21)

and the antisense primer was SEQ.ID.NO:6 (see note above regardingSEQ.ID.NOS. 6 and 11).

Two second round PCR reactions were performed separately to link the 5′and middle fragment (5′M PCR) and the middle and 3′ fragment (M3′ PCR).The 5′M PCR co-template used was the 5′ and middle PCR fragment asdescribed above, the sense primer was SEQ.ID.NO:12 and the antisenseprimer was SEQ.ID.NO:20. The 5′M PCR procedure resulted in an 857 bp PCRfragment.

The M3′ PCR used the middle and M3′ PCR fragment described above as theco-template, SEQ.ID.NO: 19 as the sense primer and SEQ.ID.NO:6 (see noteabove regarding SEQ.ID.NOS. 6 and 11) as the antisense primer, andgenerated a 784 bp amplification product. The final round of PCR wasperformed using the 857 bp and 784 bp fragments from the second roundPCR as the co-template, and SEQ.ID.NO: 12 and SEQ.ID.NO: 6 (see noteabove regarding SEQ.ID.NOS. 6 and 11) as the sense and the antisenseprimer, respectively. The 1.32 kb amplification product from the finalround of PCR was digested with Pst I and Eco RI. Then resulting 1 kb PstI-Eco RI fragment was used to replace the corresponding fragment of theendogenous human 5-HT2A to generate mutant 5-HT2A with 5-HT2C:C310K/IC3. The Apa I-Xba fragment of AP3 was used to replace thecorresponding fragment in mutant 5-HT2A with 5-HT2C: C310K/IC3 togenerate AP4.

Example 2 Receptor Expression

A. pCMV

Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous receptors discussed herein, it is most preferred that thevector utilized be pCMV. This vector was deposited with the AmericanType Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd.,Manassas, Va. 20110-2209 USA) under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure. The DNA was testedby the ATCC and determined to be viable. The ATCC has assigned thefollowing deposit number to pCMV: ATCC #203351. See FIG. 8.

B. Transfection Procedure

For the generic assay ([³⁵S]GTPγS; Example 3) and the antagonist bindingassay (mesulergine; Example 4), transfection of COS-7 or 293T cells wasaccomplished using the following protocol.

On day one, 5×10⁶ COS-7 cells or 1×10⁷ 293T cells per 150 mm plate wereplated out. On day two, two reaction tubes were prepared (theproportions to follow for each tube are per plate): tube A was preparedby mixing 20 μg DNA (e.g., pCMV vector; pCMV vector AP-1 cDNA, etc.) in1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B wasprepared by mixing 120 μl lipofectamine (Gibco BRL) in 1.2 ml serum freeDMEM. Tubes A and B were then admixed by inversions (several times),followed by incubation at room temperature for 30-45 min. The admixtureis referred to as the “transfection mixture”. Plated COS-7 cells werewashed with 1×PBS, followed by addition of 10 ml serum free DMEM. 2.4 mlof the transfection mixture was then added to the cells, followed byincubation for 4 hrs at 37° C./5% CO₂. The transfection mixture was thenremoved by aspiration, followed by the addition of 25 ml of DMEM/10%Fetal Bovine Serum. Cells were then incubated at 37° C./5% CO₂. After 72hr incubation, cells were then harvested and utilized for analysis.

Example 3 GTP Membrane Binding Scintillation Proximity Assay

The advantages of using [³⁵S]GTPγS binding to measure constitutiveactivation are that: (a) [³⁵S]GTPγS binding is generically applicable toall G protein-coupled receptors; and (b) [³⁵S]GTPγS binding is proximalat the membrane surface, thereby making it less likely to pick-upmolecules which affect the intracellular cascade. The assay utilizes theability of G protein-coupled receptors to stimulate [³⁵S]GTPγS bindingto membranes expressing the relevant receptors. Therefore, the assay maybe used to directly screen compounds at the disclosed serotoninreceptors.

FIG. 9 demonstrates the utility of a scintillation proximity assay canbe utilized to monitor the binding of [³⁵S]GTPγS to membranesexpressing, e.g., the endogenous human 5-HT2C receptor expressed in COScells. In brief, a preferred protocol for the assay is such that theassay was incubated in 20 mM HEPES, pH 7.4, binding buffer with 0.3 nM[³⁵S]GTPγS and 12.5 μg membrane protein and 1 μM GDP for 30 minutes.Wheatgerm agglutinin beads (25 μl; Amersham) were then added and themixture was incubated for another 30 minutes at room temperature. Thetubes were then centrifuged at 1500×g for 5 minutes at room temperatureand then counted in a scintillation counter. As shown in FIG. 9,serotonin, which as the endogenous ligand activates the 5-HT_(2C)receptor, stimulated [³⁵S]GTPγS binding to the membranes in aconcentration dependant manner. The stimulated binding can be completelyinhibited by 30 μM mianserin, a compound considered as a classical5-HT2C antagonist, but also known as a 5-HT2C inverse agonist.

Although this assay measures agonist-induced binding of [³⁵S]GTPγS tomembranes and can be routinely used to measure constitutive activity ofreceptors, the present cost of wheatgerm agglutinin beads may beprohibitive. A less costly but equally applicable alternative also meetsthe needs of large-scale screening. Flash plates and Wallac™scintistrips may be used to format a high throughput [³⁵S]GTPγS bindingassay. This technique allows one to monitor the tritiated ligand bindingto the receptor while simultaneously monitoring the efficacy via[³⁵S]GTPγS binding. This is possible because the Wallac™ beta countercan switch energy windows to analyze both tritium and ³⁵S-labeledprobes.

Also, this assay may be used for detecting of other types of membraneactivation events that result in receptor activation. For example, theassay may be used to monitor ³²P phosphorylation of a variety ofreceptors (including G protein-coupled and tyrosine kinase receptors).When the membranes are centrifuged to the bottom of the well, the bound[³⁵S]GTPγS or the ³²P-phosphorylated receptor will activate thescintillant coated on the wells. Use of Scinti® strips (Wallac™)demonstrate this principle. Additionally, this assay may be used formeasuring ligand binding to receptors using radiolabeled ligands. In asimilar manner, the radiolabeled bound ligand is centrifuged to thebottom of the well and activates the scintillant. The [³⁵S]GTPγS assayresults parallel the results obtained in traditional second messengerassays of receptors.

As shown in FIG. 10, serotonin stimulates the binding of [³⁵S]GTPγS tothe endogenous human 5-HT2C receptor, while mianserin inhibits thisresponse; furthermore, mianserin acts as a partial inverse agonist byinhibiting the basal constitutive binding of [³⁵S]GTPγS to membranesexpressing the endogenous human 5-HT2C receptor. As expected, there isno agonist response in the absence of GDP since there is no GDP presentto exchange for [³⁵S]GTPγS. Not only does this assay system demonstratethe response of the native %-HT2C receptor, but it also measures theconstitutive activation of other receptors.

FIG. 11A and FIG. 11B demonstrate the enhanced binding of [³⁵S]GTPγS tomembranes prepared from 293T cells expressing the control vector alone,the native human 5-HT2C receptor or the AP-1 receptor was observed (datanot shown). The total protein concentration used in the assay affectsthe total amount of [³⁵S]GTPγS binding for each receptor. The c.p.m.differential between the pCMV transfected and the constitutively activemutant receptor increased from approximately 1000 c.p.m at 10 μg/well toapproximately 6-8000 c.p.m. at 75 μg/well protein concentration, asshown in FIG. 11.

The AP-1 receptor showed the highest level of constitutive activationfollowed by the wild type receptor, which also showed enhanced[³⁵S]GTPγS binding above basal. This is consistent with the ability ofthe endogenous human 5-HT2C receptor to accumulate intracellular IP3 inthe absence of 5HT stimulation (Example 5) and is also consistent withpublished data claiming that the endogenous human 5-HT2C receptor has ahigh natural basal activity. Therefore, the AP-1 receptor demonstratesthat constitutive activity may be measured by proximal [³⁵S]GTPγSbinding events at the membrane interface.

Example 4 Serotonin Receptor Agonist/Antagonist Competitive BindingAssay

Membranes were prepared from transfected COS-7 cells (see Example 2) byhomogenization in 20 mM HEPES and 10 mM EDTA, pH 7.4 and centrifuged at49,000×g for 15 min. The pellet was resuspended in 20 mM HEPES and 0.1mM EDTA, pH 7.4, homogenized for 10 sec. using a Polytron homogenizer(Brinkman) at 5000 rpm and centrifuged at 49,000×g for 15 min. The finalpellet was resuspended in 20 mM HEPES and 10 mM MgCl₂, pH 7.4,homogenized for 10 sec. using polytron homogenizer (Brinkman) at 5000rpm.

Assays were performed in triplicate 200 μl volumes in 96 well plates.Assay buffer (20 mM HEPES and 10 mM MgCl₂, pH 7.4) was used to dilutemembranes, ³H-LSD, ³H-mesulergine, serotonin (used to definenon-specific for LSD binding) and mianserin (used to define non-specificfor mesulergine binding). Final assay concentrations consisted of 1 nM³H-LSD or 1 nM ³H-mesulergine, 50 μg membrane protein and 100 μmserotonin or mianserin. LSD assays were incubated for 1 hr at 37° C.,while mesulergine assays were incubated for 1 hr at room temperature.Assays were terminated by rapid filtration onto Wallac Filtermat Type Bwith ice cold binding buffer using Skatron cell harvester. Theradioactivity was determined in a Wallac 1205 BetaPlate counter.

Example 5 Intracellular IP3 Accumulation Assay

For the IP₃ accumulation assay, a transfection protocol different fromthe protocol set forth in Example 2 was utilized. In the followingexample, the protocols used for days 1-3 were slightly different for thedata generated for FIGS. 12 and 14 and for FIGS. 13 and 15; the protocolfor day 4 was the same for all conditions.

A. COS-7 and 293 Cells

On day one, COS-7 cells or 293 cells were plated onto 24 well plates,usually 1×10⁵ cells/well or 2×10⁵ cells/well, respectively. On day two,the cells were transfected by first mixing 0.25 ug DNA (see Example 2)in 50 μl serum-free DMEM/well and then 2 μl lipofectamine in 50 μlserum-free DMEM/well. The solutions (“transfection media”) were gentlymixed and incubated for 15-30 minutes at room temperature. The cellswere washed with 0.5 ml PBS and then 400 μl of serum free media wasmixed with the transfection media and added to the cells. The cells werethen incubated for 3-4 hours at 37° C./5% CO₂. Then the transfectionmedia was removed and replaced with 1 ml/well of regular growth media.On day 3, the media was removed and the cells were washed with 0.5 mlPBS. Then 0.5 ml inositol-free/serum-free media (GIBCO BRL) was added toeach well with 0.25 μCi of ³H-myo-inositol/well and the cells wereincubated for 16-18 hours overnight at 37° C./5% CO₂. Protocol A.

B. 293 Cells

On day one, 1×10⁷ 293 cells per 150 mm plate were plated out. On daytwo, two reaction tubes were prepared (the proportions to follow foreach tube are per plate): tube A was prepared by mixing 20 μg DNA (e.g.,pCMV vector; pCMV vector AP-1 cDNA, etc.) in 1.2 ml serum free DMEM(Irvine Scientific, Irvine, Calif.); tube B was prepared by mixing 120μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and Bwere then admixed by inversions (several times), followed by incubationat room temperature for 3045 min. The admixture is referred to as the“transfection mixture”. Plated 293 cells were washed with 1×PBS,followed by addition of 10 ml serum free DMEM. 2.4 ml of thetransfection mixture was then added to the cells, followed by incubationfor 4 hrs at 37° C./5% CO₂. On day 3, cells were trypsinized andcounted, followed by plating of 1×10⁶ cells/well (poly D-lysine treated12-well plates). Cells were permitted to adhere to the wells, followedby one wash with 1×PBS. Thereafter, 0.5 μCi ³H-inositol in 1 mlinositol-free DMEM was added per well. Protocol B.

On day 4, the cells were washed with 0.5 ml PBS and then 0.45 ml ofassay medium was added containing inositol-free/serum free media, 10 μMpargyline, 10 mM lithium chloride, or 0.4 ml of assay medium and 50 μlof 10× ketanserin (ket) to a final concentration of 10 μM. The cellswere then incubated for 30 minutes at 37° C. Then the cells were washedwith 0.5 ml PBS and 200 μl of fresh/ice cold stop solution (1M KOH; 18mM Na-borate; 3.8 mM EDTA) was added/well. The solution was kept on icefor 5-10 minutes or until the cells were lysed and then neutralized by200 μl of fresh/ice cold neutralization sol. (7.5% HCL). The lysate wasthen transferred into 1.5 ml micro-centrifuge tubes and 1 ml ofchloroform/methanol (1:2) was added/tube. The solution was vortexed for15 seconds and the upper phase was applied to a Biorad AG1-X8 anionexchange resin (100-200 mesh). The resin was washed with water and 0.9ml of the upper phase was loaded onto the column. The column was washedwith 10 ml of 5 mM myoinositol and 10 ml of 5 mM Na-borate/60 mMNa-formate. The inositol trisphosphates were eluted into scintillationvials containing 10 ml of scintillation cocktail with 2 ml of 0.1 Mformic acid/1 M ammonium formate. The columns were regenerated bywashing with 10 ml of 0.1 M formic acid/3M ammonium formate and rinsedtwice with dd H₂O and stored at room temperature in water. Results arediscussed below.

FIG. 12 is an illustration of IP3 production from the human 5-HT2Areceptor which was mutated using the same point mutation as set forth inCasey, which rendered the rat receptor constitutively active. Theresults represented in FIG. 12, support the position that when the pointmutation shown to activate the rat receptor is introduced into the humanreceptor, little activation of the receptor is obtained that would allowfor appropriate screening of candidate compounds, with the responsebeing only moderately above that of the endogenous human 5-HT2Areceptor. Generally, a response of at least 2× above that of theendogenous response is preferred.

FIG. 13 provides an illustration comparing IP3 production fromendogenous 5-HT2A receptor and the AP4 mutation. The results illustratedin FIG. 13 support the position that when the novel mutation disclosedherein is utilized, a robust response of constitutive IP3 accumulationis obtained (e.g., over 2× that of the endogenous receptor).

FIG. 14 provides an illustration of IP3 production from AP3. The resultsillustrated in FIG. 14 support the position that when the novel mutationdisclosed herein is utilized, a robust response of constitutive IP3accumulation is obtained.

FIG. 15 provides bar-graph comparisons of IP3 accumulation betweenendogenous human 5-HT2C receptor and AP-1. Note that the endogenousreceptor has a high degree of natural constitutive activity relative tothe control CMV transfected cells (i.e., the endogenous receptor appearsto be constitutively activated).

Example 6 Screening of Compounds known to have 5-HT_(2C) AntagonistActivity Against Non-Endogenous, Constitutively Activated HumanSerotonin Receptor: AP-1

A final concentration of 12.5 μg membranes prepared from COS7 cells (seeExample 2) transiently expressing constitutively active mutant human5HT2C receptor AP-1 were incubated with binding buffer (20 mM HEPES, pH7.4, 100 mM NaCl, 20 mM MgCl₂.6H, O, 0.2% saponin, and 0.2 mM ascobate),GDP(1 μM) and compound in a 96-well plate format for a period of 60minutes at ambient room temperature. Plates were then centrifuged at4,000 rpm for 15 minutes followed by aspiration of the reaction mixtureand counting for 1 minute in a Wallac™ MicroBeta plate scintillationcounter. A series of compounds known to possess reported 5HT2Cantagonist activity were determined to be active in the [³⁵S]GTPγSbinding assay using AP-1. IC₅₀ determinations were made for thesecommercially available compounds (RBI, Natick, Mass.). Results aresummarized in Table 3. For each determination, eight concentrations oftest compounds were tested in triplicate. The negative control in theseexperiments consisted of AP-1 receptor without test compound addition,and the positive control consisted of 12.5 μg/well of COS7 cellmembranes expressing the CMV promoter without expressed AP-1 receptor.

TABLE 3 IC₅₀ (nM) in GTP-γ-[³⁵S] Test Compound Known Pharmacology AssayMetergoline 5HT2/lC antagonist 32.0 Mesulergine 5HT2/lC antagonist 21.2Methysergide 5HT2/lC antagonist 6.1 Methiothepin 5HT1 antagonist 20.4Normethylclozapin 5HT2/lC antagonist 21.4 Fluoxetine 5HT reuptakeinhibitor 114.0 Ritanserin 5HT2/lC antagonist 19.4The IC₅₀ results conform that the seven tested compounds showedantagonist activity at the AP-1 receptor.

Example 7 Screening of Candidate Compounds Against Non-Endogenous,Constitutively Activated Human Serotonin Receptors: AP-1

Approximately 5,500 candidate compounds (Tripos, Inc., St. Louis, Mo.)were screened using the assay protocol of Example 3 (with AP-1 mutantreceptor) for identification as inverse agonists against the receptor;for this assay, an arbitrary cut-off of at least 50% inhibition wasestablished for identification of inverse agonists. Approximately 120 ofthese compounds evidenced at least 50% inhibition of [³⁵S]GTPγS bindingat 10 μM candidate compound (data not shown).

Example 8 Screening of Selected Compounds to Confirm Receptor Binding:AP-1

The candidate compounds identified from Example 7 were then screenedusing the assay protocol of Example 4 (mesulergine), using the AP-1mutant receptor. IC₅₀ (nM) values were determined; five of the nearly120 compounds of Example 7 were determined to have potent bindingaffinity for the receptor. Results are summarized in Table 4.

TABLE 4 IC₅₀ (nM) in Mesulergine Candidate Compound Assay 102461 205.0102788 46.5 100341 209.0 100431 147.0 103487 1,810.0

Example 9a General Screening Paradigm: Selection of Pre-ClinicalCandidate Leads

The “primary” screen designed to directly identify human5HT_(2A)/5HT_(2C) receptor inverse agonists consisted of amembrane-based GTPγS binding assay utilizing membranes prepared fromCOS7 cells transiently transfected with AP-1 human receptor. Candidatecompounds (10 μM final assay concentration) directly identified asinhibiting receptor-mediated increases in GTPγS binding by greater than50-75% (arbitrary cut-off value) were considered active “hits”. Primaryassay hits were then re-tested in the same assay to reconfirm theirinverse agonist activity. If primary assay hits were reconfirmed active(50% or greater inhibition), and therefore directly identified as, e.g.,an inverse agonist, one of two approaches were available: (a) so-called“directed libraries” could be created, i.e., additional candidatecompounds were synthesized based upon the structures of the reconfirmedhits (geared towards, e.g., improvement in the characteristics of thecompounds) whereby the directed library compounds were then evaluatedfor the ability to compete for radioligand binding to both mutant 5HT2C(AP-1) and endogenous 5HT2A receptors, or (b) the reconfirmed hits werethen evaluated for the ability to compete for radioligand binding toboth mutant 5HT2C (AP-1) and endogenous 5HT2A receptors. Thus, whenapproach (a) was used, because these directed library candidatecompounds were based upon the structures of compounds that were directlyidentified from the membrane-based GTPγS binding assay, the directedlibrary compounds were not re-tested in the membrane-based GTPγS bindingassay but rather were then confirmed via the radioligand bindinganalysis. The radioligand binding analysis tests were initiallyperformed at 10 μM test compound in triplicate and if the compoundinhibited radiolabeled binding by 50% or more, the analysis was followedby eight concentration competition curves to determine Ki values. Thelast step in secondary assay evaluation was to determine if testcompounds were capable of inhibiting AP-3 receptor-mediated accumulationof inositol phosphates (e.g., IP₃). This final assay confirms that thedirectly identified compounds retained inverse agonist properties.

Example 9b Constitutively Activated Human 5HT_(2C) Receptor (AP-1)Mediated Facilitation of GTPγS Binding to COS7 Membranes

This protocol is substantially the same as set forth above in Example 6.

Primary screening assays measuring GTPγS binding to membranes preparedfrom COS7 cells transiently transfected with human mutated 5HT2Creceptor (AP-1) were used to directly identify inverse agonists inscreening libraries (Tripos, Inc.). Candidate compound screens wereperformed in a total assay volume of 200 μl using scintillant-coatedWallac Scintistrip™ plates. The primary assay was comprised of thefollowing chemicals (at indicated final assay concentrations): 20 mMHEPES, pH 7.4, 100 mM NaCl, 20 mM MgCl₂, 0.2% saponin, 0.2 mM ascorbicacid, 1 μM GDP, 0.3 nM GTPγ³⁵S, and 12.5 μg of the above definedmembranes. Incubations were performed for 60 minutes at ambient roomtemperature. The binding assay incubation was terminated bycentrifugation of assay plates at 4,000 rpm for 15 minutes, followed byrapid aspiration of the reaction mixture and counting in a WallacMicroBeta™ scintillation counter.

Primary screening of candidate compounds initially involved testing of72 test compounds per assay plate (96-well plates were utilized), at afinal assay concentration of 10 μM candidate compound, in singlereplicates. A total of sixteen wells of each plate were dedicated for aneight concentration clozapine (a confirmed 5HT2C/2A inverse agonist)dose response curve (duplicate determinations at each concentration).Finally, a total of five assay wells of each plate were dedicated todefine the negative control (AP-1 receptor expressing membranes withoutaddition of candidate compounds) and three wells from each plate todefine the positive control (membranes without AP-1 receptor).

Reconfirmation experiments involve re-testing candidate compounds in thesame assay described above, except that candidate compounds wereevaluated in triplicate, thus allowing evaluation of 24 compounds per96-well assay plate. Similar to the primary assay plates, an eightconcentration clozapine dose response curve (duplicate determinations ateach concentration) and the same negative and positive control wellswere also included within each 96-well plate.

Example 9c(1) Competition Studies Mutated Human 5HT2C Receptor (AP-1)

Radioligand binding competition experiments were performed in a totalassay volume of 200 μl using standard 96-well microtiter plates. Thefinal assay ingredients consisted of assay buffer (20 mM HEPES and 10 mMMgCl₂), 1 nM [³H]mesulergine, and 50 μg of membranes (COS7 with AP-1 asdefined above). Nonspecific [³H]mesulergine binding was defined in thepresence of 100 μM mianserin. Incubations were performed for 1 hour at37° C. Receptor bound radioligand was resolved from free radioligand byrapid filtration of the assay mixture over a Wallac Filtermat™ Type Bfilter, followed by washing with ice-cold assay buffer using a Skatron™cell harvester. Radioactivity was counted using a Wallac 1205 BetaPlate™counter. Each assay plate contained five negative control wells(membranes expressing receptor and no candidate compound addition) andthree positive control wells (each containing 100 μM mianserin). For oneconcentration tests, candidate compounds were diluted into assay bufferand screened at a final concentration of 10 μM, in triplicate. For IC₅₀determinations, candidate compounds were diluted in assay buffer andeight different concentrations were evaluated, in triplicate. A total of16 wells were designated for an eight concentration mianserin doseresponse curve evaluation for both assays.

Example 9C(2) Competition Studies Wild Type Human 5HT2A Receptor

Radioligand binding competition experiments were performed in a totalassay volume of 200 μl using standard 96-well microtiter plates. Thefinal assay ingredients comprised assay buffer (20 mM HEPES and 10 mMMgCl₂), 1 nM [³H]LSD, and 50 μg of the above-defined membranes (COS7with AP-1). Nonspecific [³H]LSD binding was defined in the presence of100 μM serotonin. Incubations were performed for 1 hour at 37° C.Receptor bound radioligand was resolved from free radioligand by rapidfiltration of the assay mixture over a Wallac Filtermat™ Type B filter,followed by washing with ice-cold assay buffer using a Skatron™ cellharvester. Radioactivity was counted using a Wallac 1205 BetaPlate™counter. Each assay plate contained five negative control wells(membranes expressing receptor and no candidate compound addition) andthree positive control wells (containing 100 μM mianserin). For oneconcentration tests, candidate compounds were diluted into assay bufferand screened at a final concentration of 10 μM in triplicate. For IC₅₀determinations, candidate compounds were diluted in assay buffer andeight different concentrations were evaluated in triplicate. A total of16 wells were designated for an eight concentration serotonin doseresponse curve evaluation for both assays.

Example 9d Receptor-Mediated Inositol Phosphate Accumulation

Candidate compound identified in the assays of Examples 9a-9c were thenevaluated for inositol phosphate accumulation, following the protocol ofExample 5 (COS7 cells expressing human mutated 5HT2A receptor, AP-3),modified as follows: tube A was prepared by mixing 16 μg DNA (e.g., pCMVvector; pCMV vector AP-1 cDNA, etc.) in 1.0 ml serum free DMEM (IrvineScientific, Irvine, Calif.); tube B was prepared by mixing 60 μllipofectamine (Gibco BRL) in 1.0 ml serum free DMEM. Tubes A and B werethen admixed by inversions (several times), followed by incubation atroom temperature for 30 min. The admixture is referred to as the“transfection mixture”. Plated 293 cells were washed with 10 ml SerumFree DMEM, followed by addition of 11 ml Serum Free DMEM. 2.0 ml of thetransfection mixture was then added to the cells, followed by incubationfor 5 hrs at 37° C./5% CO₂. On day 3, cells were trypsinized andcounted, followed by plating of 1×10⁶ cells/well (12-well plates). Cellswere permitted to adhere to the wells for 8 hrs, followed by one washwith 1× PBS. Thereafter, 0.5 μCi ³H-inositol in 1 ml inositol-free DMEMwas added per well.

On day 4, the cells were washed with 1.5 ml PBS and then 0.9 ml of assaymedium was added containing inositol-free/serum free media, 10 μMpargyline, 10 mM lithium chloride, for 5 min in 37° C./5% CO₂ followedby 100%1 addition of candidate compound diluted in the same material.The cells were then incubated for 120 minutes at 37° C. Then the cellswere washed with 1.5 ml PBS and 200 μl of fresh/icecold stop solution(1M KOH; 18 mM Na-borate; 3.8 mM EDTA) was added/well. The solution waskept on ice for 5-10 minutes or until the cells were lysed and thenneutralized by 200 μl of fresh/ice cold neutralization sol. (7.5% HCL).The lysate was then transferred into 1.5 ml micro-centrifuge tubes and 1ml of chloroform/methanol (1:2) was added/tube. The solution wasvortexed for 15 seconds and the upper phase was applied to a BioradAG1-X8 anion exchange resin (100-200 mesh). The resin was washed withwater and 0.9 ml of the upper phase was loaded onto the column. Thecolumn was washed with 10 ml of 5 mM myoinositol and 10 ml of 5 mMNa-borate/60 mM Na-formate. The inositol trisphosphates were eluted intoscintillation vials containing 10 ml of scintillation cocktail with 2 mlof 0.1 M formic acid/1 M ammonium formate. The columns were regeneratedby washing with 10 ml of 0.1 M formic acid/3M ammonium formate andrinsed twice with ddH₂O and stored at room temperature in water.

Following this round of assaying, candidate compounds having an IC₅₀value of less than 10 μM were considered as potential leads for thedevelopment of pharmaceutical compositions.

Screening Candidate Compounds

Following the protocols set forth above, one compound, 103487 (Example8, supra) evidenced the following results:

GTPγS AP-1 GTPγS AP-1 Competitive Competitive Inositol PercentInhibition Percent Inhibition Binding AP-1 Binding WT 5HT2A PhosphateFIG. Relative To Positive Relative To Positive ([³H]mesulergine)([³H]LSD) Accumulation AP-3 No. Control (Primary) Control (Reconfirm)IC₅₀ Value (nM) IC₅₀ Value (nM) IC₅₀ Value (nM) 15A −1% 31% 2100 46 52(103487) 850 90

Based upon these results, structure activity analysis of the 103487compound suggested that a series of derivatives of3-(4-bromo-1-methylpyrazole-3-yl)phenylamine would exhibit similar5-HT2A activity and selectivity. A series of derivatives of3-(4-bromo-1-methylpyrazole-3-yl)phenylamine were synthesized. These“directed” library compounds (Tripos, Inc.) were then analyzed inaccordance with the protocols of Examples 9c(1), 9c(2) and 9d.

This series of compounds exhibits highly selective 5-HT_(2A) activity.Accordingly, in the first aspect of the invention, a series of compoundspossessing 5-HT_(2A) receptor activity that are useful as inverseagonists at such receptors is designated by the general formula (A):

Wherein:

-   W is lower alkyl (C₁₋₆), or halogen;-   V is lower alkyl (C₁₋₆), or halogen;-   X is either Oxygen or Sulfur;-   Y is NR²R³, or (CH₂)_(m)R⁴, or O(CH₂)_(n)R⁴;-   Z is lower alkyl (C₁₋₆);-   m=0-4-   n=0-4-   R¹ is H or lower alkyl(C₁₋₄);-   R² is H or lower alkyl(C₁₋₄);

R³ and R⁴ are independently a C₁₋₆ alkyl, or C₂₋₆ alkenyl, orcycloalkyl, or aryl group and each said group may be optionallysubstituted by up to four substituents in any position independentlyselected from CF₃, CCl₃, NO₂, OH, CONR⁵R⁶, NR⁵R⁶, OCF₃, SMe, COOR⁷,SO₂NR⁵R⁶, SO₃R⁷, CO-lower alkyl (for example, COMe and COEt), SCF₃CN,C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy (for example, OMe and OEt), C₃₋₆cycloalkyl, C₁₋₆ alkyl (for example Me), aryl, and aryloxy wherein eachof the C₃₋₆ cycloalkyl, C-6 alkyl, aryl, or aryloxy groups may befurther optionally substituted by up to four substituents in anyposition independently selected from CF₃, CCl₃, NO₂, OH, CONR⁵R⁶, NR⁵R⁶,NHCOCH₃, OCF₃, SMe, COOR⁷, SO₃R⁷, SO₂NR⁵R⁶, CO-lower alkyl (for exampleCOMe and COEt), SCF₃CN, C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy (forexample, OMe and OEt), C₃₋₆ cycloalkyl, C₁₋₆ alkyl, and aryl;

R⁵ and R⁶ are independently a H, or C₁₋₆ alkyl, or C₂₋₆ alkenyl, orcycloalkyl, or aryl, or CH₂ aryl group and each said group may beoptionally substituted by up to four substituents in any positionindependently selected from CF₃, CCl₃, NO₂, OH, CONR⁷R⁸, NR⁷R⁸, NHCOCH₃,OCF₃, SMe, COOR⁹, SO₃R⁷, SO₂NR⁷R⁸, CO-lower alkyl (for example, COMe andCOEt), SCF₃, CN, C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy (for example,OMe and OEt), C₃₋₆ cycloalkyl, C-6 alkyl, and aryl wherein each of theC₃₋₆ cycloalkyl, C₁₋₆ alkyl, or aryl groups may be further optionallysubstituted by up to four substituents in any position independentlyselected from CF₃, CCl₃, NO₂, OH, CONR⁸R⁹, N R⁸R⁹, NHCOCH₃, OCF₃, SMe,COOR⁷, SO₂NR⁸R⁹, SO₃R⁷, CO-lower alkyl (for example, COMe and COEt),SCF₃, CN, C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy (for example, OMe andOEt), C₃₋₆ cycloalkyl, C₁₋₆ alkyl, and aryl,

or R⁵ and R⁶ may form part of a 5, 6 or 7 membered cyclic structurewhich may be either saturated or unsaturated and that may contain up tofour heteroatoms selected from O, N or S and said cyclic structure maybe optionally substituted by up to four substituents in any positionindependently selected from CF₃, CCl₃, Me, NO₂, OH, OMe, OEt, OCF₃, SMe,COOR⁷, SO₂NR⁸R⁹, SO₃R⁷, HCOCH₃, COEt, COMe, or halogen;

R⁷ may be independently selected from H or C₁₋₆ alkyl;

R⁸ and R⁹ are independently a H, or C₁₋₆ alkyl, or C₂₋₆ alkenyl, orcycloalkyl, or aryl, or CH₂ aryl group and each said group may beoptionally substituted by up to four substituents in any positionindependently selected from halogen, CF₃, OCF₃, OEt, CCl₃, Me, NO₂, OH,OMe, SMe, COMe, CN, COOR⁷, SO₃R⁷, COEt, NHCOCH₃, or aryl;

an aryl moiety can be a 5 or 6 membered aromatic hetero-cyclic ring(containing up to 4 hetero atoms independently selected from N, O, or S)or a 6 membered aromatic non-heterocyclic ring or a polycycle;

C₁₋₆ alkyl moieties can be straight chain or branched;

optionally substituted C₁₋₆ alkyl moieties can be straight chain orbranched;

C₂₋₆ alkenyl moieties can be straight chain or branched; and

optionally substituted C₂₋₆ alkenyl moieties can be straight chain orbranched.

Examples of suitable C₁₋₆ alkyl groups include but are not limited tomethyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl.

Halogens are typically F, Cl, Br, and I.

Examples of 5 or 6 membered ring moieties include, but are notrestricted to, phenyl, furanyl, thienyl, imidazolyl, pyridyl, pyrrolyl,oxazolyl, isoxazolyl, triazolyl, pyrazolyl, tetrazolyl, thiazolyl andisothiazoyl. Examples of polycycle moieties include, but are notrestricted to, naphthyl, benzothiazolyl, benzofuranyl, benzimidazolyl,quinolyl, isoquinolyl, indolyl, quinoxalinyl, quinazolinyl andbenzothienyl.

A more preferred series of compounds possessing 5-HT_(2A) receptoractivity that are useful as inverse agonists at such receptors isdesignated by the general formula (B):

Wherein:

W is Me, or Et, or halogen;

X is either Oxygen or Sulfur;

Y is NR²R³, or (CH₂)_(m)R⁴, or O(CH₂)_(n)R⁴;

Z is lower alkyl (C₁₋₆);

m=0-4

n=0-4

R¹ is H or lower alkyl (C₁₋₄);

R² is H or lower alkyl(C₁₋₄);

R³ and R⁴ are independently a C-6 alkyl, or C₂₋₆ alkenyl, or cycloalkyl,or aryl group and each said group may be optionally substituted by up tofour substituents in any position independently selected from CF₃, CCl₃,NO₂, OH, CONR⁵R⁶, NR⁵R⁶, OCF₃, SMe, COOR⁷, SO₂NR⁵R⁶, SO₃R⁷, CO-loweralkyl, SCF₃CN, C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy, C₃₋₆ cycloalkyl,C₁₋₆ alkyl, aryl, and aryloxy wherein each of the C₃₋₆ cycloalkyl, C₁₋₆alkyl, aryl, or aryloxy groups may be further optionally substituted byup to four substituents in any position independently selected from CF₃,CCl₃, NO₂, OH, CONR⁵R⁶, NR⁵R⁶, NHCOCH₃, OCF₃, SMe, COOR⁷, SO₃R⁷,SO₂NR⁵R⁶, CO-lower alkyl, SCF₃CN, C₂₋₆ alkenyl, H, halogens, C₁₋₄alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, and aryl;

R⁵ and R⁶ are independently a H, or C₁₋₆ alkyl, or C₂₋₆ alkenyl, orcycloalkyl, or aryl, or CH, aryl group and each said group may beoptionally substituted by up to four substituents in any positionindependently selected from CF₃, CCl₃, NO₂, OH, CONR⁷R⁸, NR⁷R⁸, NHCOCH₃,OCF₃, SMe, COOR⁹, SO₃R⁷, SO₂NR⁷R⁸, CO-lower alkyl, SCF₃, CN, C₂₋₆alkenyl, H, halogens, C₁₋₄ alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, and arylwherein each of the C₃₋₆ cycloalkyl, C₁₋₆ alkyl, or aryl groups may befurther optionally substituted by up to four substituents in anyposition independently selected from CF₃ CCl₃, NO₂, OH, CONR⁸R⁹, NR⁸R⁹,NHCOCH₃, OCF₃, SMe, COOR⁷, SO₂NR⁸R⁹, SO₃R⁷, CO-lower alkyl, SCF₃, CN,C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, andaryl,

or R⁵ and R⁶ may form part of a 5, 6 or 7 membered cyclic structurewhich may be either saturated or unsaturated and that may contain up tofour heteroatoms selected from O, N or S and said cyclic structure maybe optionally substituted by up to four substituents in any positionindependently selected from CF₃, CCl₃, Me, NO₂, OH, OMe, OEt, OCF₃, SMe,COOR⁷, SO₂NR⁸R⁹, SO₃R⁷, NHCOCH₃, COEt, COMe, or halogen;

R⁷ may be independently selected from H or C₁₋₆ alkyl;

R⁸ and R⁹ are independently a H, or C₁₋₆ alkyl, or C₂₋₆ alkenyl, orcycloalkyl, or aryl, or CH₂aryl group and each said group may beoptionally substituted by up to four substituents in any positionindependently selected from halogen, CF₃, OCF₃, OEt, CCl₃, Me, NO₂, OH,OMe, SMe, COMe, CN, COOR⁷, SO₃R⁷, COEt, NHCOCH₃, or aryl;

an aryl moiety can be a 5 or 6 membered aromatic hetero-cyclic ring(containing up to 4 hetero atoms independently selected from N, O, or S)or a 6 membered aromatic non-heterocyclic ring or a polycycle;

C₁₋₆ alkyl moieties can be straight chain or branched;

optionally substituted C₁₋₆ alkyl moieties can be straight chain orbranched:

C₂₋₆ alkenyl moieties can be straight chain or branched; and

optionally substituted C₂₋₆ alkenyl moieties can be straight chain orbranched.

Examples of suitable C₁₋₆ alkyl groups include but are not limited tomethyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl.

Halogens are typically F, Cl, Br, and I.

Examples of 5 or 6 membered ring moieties include, but are notrestricted to, phenyl, furanyl, thienyl, imidazolyl, pyridyl, pyrrolyl,oxazolyl, isoxazolyl, triazolyl, pyrazolyl, tetrazolyl, thiazolyl andisothiazoyl. Examples of polycycle moieties include, but are notrestricted to, naphthyl, benzothiazolyl, benzofuranyl, benzimidazolyl,quinolyl, isoquinolyl, indolyl, quinoxalinyl, quinazolinyl andbenzothienyl.

A first series of compounds having 5-HT2A receptor activity isrepresented by a class (I) of compounds of formula (B) wherein Y=NR²R³:

Wherein:

Preferably R¹ and R² are H.

Preferably W is Br.

Preferably X is O.

Preferably Z is Me.

Preferably R³ is 4-trifluoromethoxyphenyl or 4-trifluoromethoxybenzyl.

Preferred compounds are:

103487N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{(4-trifluoromethoxy)phenyl}amino]carboxamide

116115 N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{(4-trifluoromethoxy)phenyl)methyl}amino]carboxamide

These two compounds demonstrated the following activities using theassay protocols defined in the Examples above:

Competitive Competitive Inositol Binding Binding Phosphate AP-1 WT5HT_(2A) Accumulation ([³H]mesulergine) ([³H]LSD) AP-3 IC₅₀ Value IC₅₀Value IC₅₀ Value Compound Number (μM) (μM) (μM) 103487 2.1 .046 .052116115 1.2 .45 .0171

Additional compounds of formula (B) wherein Y═NR²R³ are set forth below.Inositol phosphate accumulation assays evidence the activity of testcompounds. Both single concentration percentages of control values andIC₅₀ determinations indicate activity. In the tables below the columnlegends have the following meanings:

IP₃% Control: The values in this column reflect an IP Accumulation Assaywhere the test compounds were evaluated at one concentration of 10 μM.For these assays, the compound was diluted into inositol-free Dulbecco'sEagle Media containing 10 μM pargyline and 10 mM LiCl and tested at afinal assay concentration of 10 μM, in triplicate. The percent controlvalue was calculated based on the control in which no test compound wasadded.

IP₃ AP-3 IC₅₀ nM: The values in this column reflect an IP accumulationassay in which the test compound was evaluated at several differentconcentrations whereby an IC₅₀ could be determined. This columncorresponds to the column appearing in the tables above which islabeled: Inositol Phosphate Accumulation, AP-3, IC₅₀ Value (μM).

WT 5HT_(2A) LSD IC₅₀ nM: The values in this column reflect a competitivebinding assay using LSD. This column corresponds to the column appearingin the tables above which is labeled: Competitive Binding, WT 5HT_(2A),([³H]LSD), IC₅₀ Value (μM).

Compounds listed in each of the following tables reference thestructures immediately preceding the table. A “dash” in the tableindicates that no value was determined.

IP₃ IP₃ WT 5HT_(2A) % of AP-3 LSD Compound No. R¹ R² R³ R⁴ X U ControlIC₅₀ nM IC₅₀ nMN-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][(4-methylthiophenyl)amino]carboxamide116079 SCH₃ H H H O NH 16 17 4N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][(4-chlorophenyl)amino]carboxamide116081 Cl H H H O NH 10 3.2 11{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(4-fluorophenyl)carboxamide116082 F H H H O NH 11 — 7{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-[2-(trifluoromethoxy)phenyl]carboxamide116087 H H CF₃O H O NH 11 — 200{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(2-nitrophenyl)carboxamide116089 H H NO₂ H O NH 27 — 238{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(4-methoxyphenyl)carboxamide116091 MeO H H H O NH 12 — 19{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(2-methylphenyl)carboxamide116092 H H Me H O NH 32 — 131{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-[4-trifluoromethyl)phenyl]carboxamide116097 CF₃ H H H O NH 11 — 65{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(3-chlorophenyl)carboxamide116105 H Cl H H O NH 11 — 39{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-[2-chlorophenyl)carboxamide116108 H H Cl H O NH 6 — 249{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-[4-(methylethyl)phenyl]carboxamide116110 isopropyl H H H O NH 7 — 338{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(3-methoxyphenyl)carboxamide116111 H MeO H H O NH 7 — 106[{3-(4-bromo-1-methylpyrazol-3-yl)phenyl}-amino]-N-(3-methylphenyl)carboxamide116112 H Me H H O NH 14 — 57[{3-(4-bromo-1-methylpyrazol-3-yl)phenyl}-amino]-N-methyl-N-[4-(trifluoromethoxy)phenyl]carboxamide 116113 CF₃O H H H O NCH₃ — 193 2N-[4-(tert-butyl)phenyl]{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}carboxamide116119 t-butyl H H H O NH 17 — 476N-[4-(dimethylamino)phenyl]{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}carboxamide116122 NMe₂ H H H O NH 9 — 309N-(3,5-dichloro-4-methylphenyl){[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}carboxamide116138 Me Cl H Cl O NH 23 — 122{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-[4-(trifluoromethylthio)phenyl]carboxamide116139 CF₃S H H H O NH 12 — 56{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(2-fluorophenyl)carboxamide116144 H H F H O NH 12 — 372-({[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}carbonylamino)benzamide116145 H H CONH₂ H O NH 31 — 7473{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(4-cyanophenyl)carboxamide116147 CN H H H O NH 12 — 2{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(2-cyanophenyl)carboxamide116148 H H CN H O NH 30 — 348

Compound No. IP₃ AP-3 IC₅₀ nM WT 5HT_(2A) LSD IC₅₀ nMN-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-[cyclohexylamino]carboxamide116141 114 81

Compound No. R¹ R² R³ R⁴ R⁵ IP₃ AP-3 IC₅₀ nM WT 5HT_(2A) LSD IC₅₀ nMN-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-[phenylmethylamino]carboxamide116143 H H H H H 120 47N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-[{(4-fluorophenyl)methyl}amino]carboxamide116182 F H H H H 89 132N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-[{(3,4-dimethoxyphenyl)methyl}amino]-carboxamide116183 OMe OMe H H H — 1010N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-[{(3,4,5-trimethoxyphenyl)methyl}amino]-carboxamide 116184 OMe OMe H Ome H — 2960N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{(2-methylphenyl)methyl}amino]carboxamide116185 H H Me H H — 769N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{(4-methoxyphenyl)methyl}amino]carboxamide116189 OMe H H H H — 102

Compound No. R¹ R² R³ R⁴ R⁵ IP₃ AP-3 IC₅₀ nM WT 5HT_(2A) LSD IC₅₀ nMN-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{2-(4-methoxyphenyl)ethyl}amino]carboxamide116194 OMe H H H H 32 61

A second series of compounds having 5-HT_(2A) receptor activity isrepresented by a class (II) of compounds of formula (B) whereinY=O(CH₂)_(n)R4.

Wherein:

Preferably R¹ is H.

Preferably W is Br.

Preferably X is O.

Preferably Z is Me.

Preferably when n=O, R⁴ is 4-methoxyphenyl or tertiary butyl.

Preferred compounds are:

116100N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][4-methoxyphenoxy]carboxamide

116192{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}-N-(1,1-dimethylethoxy)carboxamide

These two compounds demonstrated the following activity:

Competitive Competitive Inositol Binding Binding Phosphate AP-1 WT5HT_(2A) Accumulation ([³H]mesulergine) ([³H]LSD) AP-3 IC₅₀ Value IC₅₀Value IC₅₀ Value Compound Number (μM) (μM) (μM) 116100 1.8 <0.001 0.0003116192 — 0.014 0.057

In addition to the assays discussed above, the specific activity of116100 at the 5HT_(2A) receptor was further confirmed by the following:

In Vitro Binding of 5HT_(2A) Receptor

Animals:

Animals (Sprague-Dawley rats) were sacrificed and brains were rapidlydissected and frozen in isopentane maintained at −42° C. Horizontalsections were prepared on a cryostat and maintained at −20° C.

LSD Displacement Protocol:

Lysergic acid diethylamide (LSD) is a potent 5HT2A receptor and dopamineD2 receptor ligand. An indication of the selectivity of compounds foreither or both of these receptors involves displacement ofradiolabeled-bound LSD from pre-treated brain sections. For thesestudies, radiolabeled I¹²⁵-LSD (NEN Life Sciences, Boston, Mass.,Catalogue number NEX-199) was utilized; spiperone (RBI, Natick, Mass.Catalogue number s-128) a 5HT2A receptor and dopamine D2 receptorantagonist, was also utilized. Buffer consisted of 50 nanomolarTRIS-HCl, pH 7.4.

Brain sections were incubated in (a) Buffer plus 1 nanomolar I¹²⁵-LSD;(b) Buffer plus 1 nanomolar I¹²⁵-LSD and 1 micromolar spiperone; orBuffer plus 1 nanomolar I¹²⁵-LSD and 1 micromolar 116100 for 30 minutesat room temperature. Sections were then washed 2×10 minutes at 4° C. inBuffer, followed by 20 seconds in distilled H₂O. Slides were thenair-dried.

After drying, sections were apposed to x-ray film (Kodak Hyperfilm) andexposed for 4 days.

Analysis:

FIGS. 16A-C provide representative autoradiographic sections from thisstudy. FIG. 16A evidences darker bands (derived from I¹²⁵-LSD binding)primarily in both the fourth layer of the cerebral cortex (primarily5HT_(2A) receptors), and the caudate nucleus (primarily dopamine D2receptors and some 5HT_(2A) receptors). As can be seen from FIG. 16B,spiperone, which is a 5HT_(2A) and dopamine D2 antagonist, displaces theI¹²⁵-LSD from these receptors on both the cortex and the caudate. As canbe further seen from FIG. 16C, 116100 appears to selectively displacethe I¹²⁵-LSD from the cortex (5HT_(2A)) and not the caudate (dopamineD2).

A third series of compounds having 5-HT_(2A) receptor activity isrepresented by a class (III) of compounds of formula (B) whereinY=(CH₂)_(m)R⁴:

Wherein

-   -   Preferably W is Br.    -   Preferably X is O.    -   Preferably Z is Me.    -   Preferably R¹ is H.

Preferably when m=O, R⁴ is preferably 4-trifluoromethoxyphenyl, orthiophene, or 4-chlorophenyl.

Preferred compounds are:

116101 m=0, R¹=H, R⁴=4-trifluoromethoxyphenylN-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][4-trifluoromethoxyphenyl]carboxamide

116102 m=0, R¹=H, R⁴=thiopheneN-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][2-thienyl]carboxamide

116120 m=0, R¹=H, R⁴=chlorophenylN-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][4-chloro-phenyl]carboxamide

These three compounds demonstrated the following activities:

Competitive Competitive Inositol Binding Binding Phosphate AP-1 WT5HT_(2A) Accumulation ([³H]mesulergine) ([³H]LSD) AP-3 IC₅₀ Value IC₅₀Value IC₅₀ Value Compound Number (μM) (μM) (μM) 116101 6.1 .46 0.0213116102 2.8 .17 0.080 116120 1.2 .21 0.0315

In Vivo Analysis of Compound 116102

In addition to the in vitro assays shown in the above table, the in vivoresponse of animals to the 116102 compound is demonstrated by thefollowing.

A 5HT_(2A) receptor antagonist or inverse agonist is expected todecrease amphetamine-stimulated locomotion without affecting baselinelocomotion. See, for example, Soresnon, et al, 266(2) J. Pharmacol. Exp.Ther. 684 (1993). Based upon the foregoing information, Compound 116102is a potent inverse agonist at the human 5HT2A receptor. For thefollowing study, the following parameters and protocol were utilized:

Animals, Vehicle

Adult male Sprague-Dawley rats were utilized for these studies. Animalswere housed in groups of 2-3 in hanging plastic cages with food andwater available at all times. Animals were weighed and handled for atleast one day prior to surgery and throughout the studies. For thesestudies, Vehicle consisted of 90% ethanol (100%) and 10% water.

Amphetamine-Stimulated Locomotor Activity: Assessment and Apparatus

A San Diego Instruments Flex Field apparatus was used to quantifybaseline and amphetamine-stimulated locomotor activity. This apparatusconsists of four 16″×16″ clear plastic open fields. Photocell arrays (16in each dimension) interfaced with a personal computer to automaticallyquantify activity. Several measures of activity can be assessed with theapparatus, including total photocell beam breaks. Animals (vehiclecontrol and Compound treated) were injected s.c. 30 minutes prior toinitiation of analysis. Following this 30 minute period, animals wereplaced individually into an open field and baseline activity wasassessed for 30 minutes (habituation phase). Following baseline, animalswere removed, injected with d-amphetamine sulfate (1.0 mg/kg) andimmediately returned to the open field for 150 minutes, in order tofollow the time course (10 minute intervals) of amphetamine-stimulatedlocomotor activity.

Dosing

Vehicle Control Compound 116102 Dose (mg/kg) 6 animals 6 animals 0.1 6animals 1.0 6 animals 5.0 6 animals 10.0Analysis

Results, based upon the number of recorded photobeam breaks(mean.+/−sem), are presented in FIGS. 17A-C. As supported by FIGS. 17A,Band C, a general “inverted U” shaped pattern was observed (see,generally, Sahgal, A. “Practical behavioural neuroscience: problems,pitfalls and suggestions” pp 1-8, 5 in Behavioral Neuroscience: APractical Approach, Volume 1 A. Sahgal (Ed.) 1993, IRL Press, New York).As FIG. 17 also indicates, with exception of the highest dose (10mg/kg), in vivo, the tested doses of Compound 116102 evidenced adecrease in the amphetamine-stimulated locomotion, consistent with a5HT2A receptor antagonist or inverse agonist.

Additional compounds of formula (B) wherein Y=(CH₂)_(m) R⁴ are set forthbelow.

WT IP₃ 5HT_(2A) AP-3 LSD Compound IC₅₀ IC₅₀ Name No. R¹ R² R³ R⁴ nM nMN-[3-(4-bromo-1-methylpyrazol-3- 116137 OCF₃ H H H — 106yl)phenyl]-2-[4-(trifluoromethoxy)- phenyl]acetamideN-[3-(4-bromo-1-methylpyrazol-3- 116174 H F H H 153 318 yl)phenyl]-2-(3-fluorophenyl)acetamide N-[3-(4-bromo-1-methylpyrazol-3- 116175 H OMe H H108 625 yl)phenyl]-2-(3- methoxyphenyl)acetamideN-[3-(4-bromo-1-methylpyrazol-3- 116176 H H F H 129 662 yl)phenyl]-2-(2-fluorophenyl)acetamide N-[3-(4-bromo-1-methylpyrazol-3- 116177 NO₂ H H H61 108 yl)phenyl]-2-(4- nitrophenyl)acetamide compound names notprovided 116178 H H OMe H 165 2300

Based upon the discovery of the specific inverse agonist activity of theabove identified compounds at the 5HT_(2A) receptor, a novel class ofcompounds has been identified which exhibits said activity. Accordingly,in the second aspect of the invention, there is provided a novelcompound of formula (C):

Wherein:

W is Me, or Et, or halogen;

X is either Oxygen or Sulfur;

Y is NR²R³, or (CH2)_(m) R⁴, or O(CH₂)_(n)R⁴;

Z is lower alkyl (C₁₋₆);

m=0-4;

n=0-4;

R¹ is H or lower alkyl (C₁₋₄);

R² is H or lower alkyl(C₁₋₄);

R³ is a C₁₋₆ alkyl, or C₂₋₆ alkenyl, or cycloalkyl, or (CH₂)_(k) arylgroup (k=1-4), preferably k=1, and each said group may be optionallysubstituted by up to four substituents in any position independentlyselected from CF₃, CCl₃, NO₂, OH, CONR⁵R⁶, NR⁵R⁶, OCF₃, SMe, COOR⁷,SO₂NR⁵R⁶, SO₃R⁷, CO-lower alkyl, SCF₃ CN, C₂₋₆ alkenyl, H, halogens,C₁₋₄ alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, aryl, and aryloxy wherein eachof the C₃₋₆ cycloalkyl, C₁₋₆ alkyl, aryl, or aryloxy groups may befurther optionally substituted by up to four substituents in anyposition independently selected from CF₃, CCl₃, NO₂, OH, CONR⁵R⁶, NR⁵R⁶,NHCOCH₃, OCF3, SMe, COOR⁷, SO₃R⁷, SO₂NR⁵R⁶, CO-lower alkyl, SCF₃, CN,C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, andaryl;

R⁴ is a C₁₋₆ alkyl, or C₂₋₆ alkenyl, or cycloalkyl, or aryl group andeach said group may be optionally substituted by up to four substituentsin any position independently selected from CF₃, CCl₃, NO₂, OH, CONR⁵R⁶,NR⁵R⁶, OCF₃, SMe, COOR⁷, SO₂NR⁵R⁶, SO₃R⁷, CO-lower alkyl, SCF₃ CN, C₂₋₆alkenyl, H, halogens, C₁₋₄ alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, aryl,and aryloxy wherein each of the C₃₋₆ cycloalkyl, C₁₋₆ alkyl, aryl, oraryloxy groups may be further optionally substituted by up to foursubstituents in any position independently selected from CF₃, CCl₃, NO₂,OH, CONR⁵R⁶, NR⁵R⁶, NHCOCH₃, OCF3, SMe, COOR⁷, SO₃R⁷, SO₂NR⁵R⁶, CO-loweralkyl, SCF₃, CN, C₂₋₆ alkenyl, H, halogens, CIA alkoxy, C₃₋₆ cycloalkyl,C₁₋₆ alkyl, and aryl;

R⁵ and R⁶ are independently a H, or C₁₋₆ alkyl, or C₂₋₆ alkenyl, orcycloalkyl, or aryl, or CH₂ aryl group and each said group may beoptionally substituted by up to four substituents in any positionindependently selected from CF₃, CCl₃, NO₂, OH, CONR⁷R⁸, NR⁷R⁸, NHCOCH₃,OCF₃, SMe, COOR⁹, SO₃R⁷, SO₂NR⁷R⁸, CO-lower alkyl, SCF₃, CN, C₂₋₆alkenyl, H, halogens, CIA alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, and arylwherein each of the C₃₋₆ cycloalkyl, C₁₋₆ alkyl, or aryl groups may befurther optionally substituted by up to four substituents in anyposition independently selected from CF₃, CCl₃, NO₂, OH, CONR⁸R⁹, NR⁸R⁹,NHCOCH₃, OCF₃, SMe, COOR⁷, SO₂NR⁸R⁹, SO₃R⁷, CO-lower alkyl, SCF₃, CN,C₂₋₆ alkenyl, H, halogens, C₁₋₄ alkoxy, C₃₋₆ cycloalkyl, C₁₋₆ alkyl, andaryl,

or R⁵ and R⁶ may form part of a 5, 6 or 7 membered cyclic structurewhich may be either saturated or unsaturated and that may contain up tofour heteroatoms selected from O, N or S and said cyclic structure maybe optionally substituted by up to four substituents in any positionindependently selected from CF₃, CCl₃, Me, NO₂, OH, OMe, OEt, OCF₃, SMe,COOR⁷, SO₂NR⁸R⁹, SO₃R⁷, NHCOCH₃, COEt, COMe, or halogen;

R⁷ may be independently selected from H or C₁₋₆ alkyl;

R⁸ and R⁹ are independently a H, or C₁₋₆ alkyl, or C₂₋₆ alkenyl, orcycloalkyl, or aryl, or CH₂ aryl group and each said group may beoptionally substituted by up to four substituents in any positionindependently selected from halogen, CF₃, OCF3, OEt, CCl₃, Me, NO₂, OH,OMe, SMe, COMe, CN, COOR⁷, SO₃R⁷, COEt, NHCOCH₃, or aryl;

an aryl moiety can be a 5 or 6 membered aromatic heterocyclic ring(containing up to 4 hetero atoms independently selected from N, O, or S)or a 6 membered aromatic non-heterocyclic ring or a polycycle;

C₁₋₆ alkyl moieties can be straight chain or branched;

optionally substituted C₁₋₆ alkyl moieties can be straight chain orbranched;

C₂₋₄ alkenyl moieties can be straight chain or branched; and

optionally substituted C₂₋₆ alkenyl moieties can be straight chain orbranched with the proviso that said compound is not:

-   N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][methylamino]carboxamide,    or-   N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{(4-trifluoromethoxy)phenyl}amino]carboxamide,    or-   N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][2-chlorophenyl]carboxamide,    or-   N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][2-chloro-3-pyridyl]carboxamide,    or-   N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][trichloromethyl]carboxamide.

Examples of suitable C₁₋₄ alkyl groups include but are not limited tomethyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl.

Halogens are typically F, Cl, Br, and I.

Examples of 5 or 6 membered ring moieties include, but are notrestricted to, phenyl, furanyl, thienyl, imidazolyl, pyridyl, pyrrolyl,oxazolyl, isoxazolyl, triazolyl, pyrazolyl, tetrazolyl, thiazolyl andisothiazolyl. Examples of polycycle moieties include, but are notrestricted to, naphthyl, benzothiazolyl, benzofuranyl, benzimidazolyl,quinolyl, isoquinolyl, indolyl, quinoxalinyl, quinazolinyl andbenzothienyl.

Synthetic Approaches

The compounds disclosed in this invention may be readily preparedaccording to a variety of synthetic manipulations, all of which would befamiliar to one skilled in the art. In the general syntheses set forthbelow, the labeled substituents have the same identifications as set outin the definitions of the compounds above.

Compounds of general formula (I) can be obtained via a variety ofsynthetic routes all of which would be familiar to one skilled in theart. The reaction of isocyanates with amines is a commonly practicedmethod for the formation of ureas (see Org. Syn. Coll. Vol. V, (1973),555). Amine (IV), 3-(4-bromo-2-methylpyrazole-3-yl)phenylamine,commercially available from Maybridge Chemical Company, Catalog No.KM01978, CAS No. 175201-77-1] reacts readily with isocyanates (V) ininert solvents such as halocarbons to yield the desired ureas of generalformula (I) wherein R¹=R²=H:

Alternatively the amine (IV) can be converted to the correspondingisocyanate (VI) by the action of phosgene or a suitable phosgeneequivalent, e.g. triphosgene, in an inert solvent such as a halocarbonin the presence of an organic base such as triethylamine orethyldiisopropylamine. Isocyanate (VI) reacts with amines of generalformula (VII), in an analogous fashion to that described above for thereaction of (IV) with (V), yielding the desired ureas of general formula(I) wherein R¹=H:

Alternatively wherein the isocyanate of general formula (V) is notcommercially available it can be prepared from the corresponding amineof general formula (VIII) in an analogous procedure to that describedabove for the preparation of (VI). Reaction of these isocyanates with(IV) would again yield the requisite ureas of general formula (I)wherein R¹=R²=H:

Amines of general formula (VII) are also readily converted to activatedisocyanate equivalents of general formula (IX) by the sequential actionof carbonyldiimidazole and methyl iodide in tetrahydrofuran andacetonitrile respectively (R. A. Batey et al, Tetrahedron Lett., (1998),39, 6267-6270.) Reaction of (IX) with (IV) in an inert solvent such as ahalocarbon would yield the requisite ureas of general formula (I)wherein R¹=H:

Amine (IV) may be monomethylated according to the procedure of J.Barluenga et al, J. Chem. Soc., Chem. Commun., (1984), 20, 1334-1335, oralkylated according to the procedure of P. Marchini et al, J. Org.Chem., (1975), 40(23), 3453-3456, to yield compounds of general formula(X) wherein R¹=lower alkyl. These materials may be reacted as above withreagents of general formula (V) and (IX) as depicted below:

Compounds of general formula (II) can similarly be obtained via avariety of synthetic manipulations, all of which would be familiar toone skilled in the art. The reaction of amine (IV) with chloroformates(see Org. Syn. Coll. Vol. IV, (1963), 780) of general formula (XI) in aninert solvent such as ether or halocarbon in the presence of a tertiarybase such as triethylamine or ethylisopropylamine readily yields therequisite carbamates of general formula (II) wherein R³=H. Analagouolsy,amines of general formula (X) react similarly with chloroformates (XI)to yield the requisite carbamates of general formula (II) whereinR³=lower alkyl:

An alternate route employs the ready reaction of an alcohol with anisocyanate. Thus isocyanate (VI) described previously reacts readilywith alcohols (XII) in an aprotic solvent such as ether or chlorocarbonto yield the desired carbamates of general formula (II) wherein R¹=H:

Chloroformates of general formula (XI) not commercially available may bereadily prepared from the corresponding alcohol (XII) in an inertsolvent such as toluene, chlorocarbon or ether by the action of excessphosgene (see Org. Syn. Coll. Vol. III, (1955), 167):

Compounds of general formula (III) can be obtained via a variety ofsynthetic manipulations, all of which would be familiar to one skilledin the art. The reaction of amine (IV) with acid chlorides (see Org.Syn. Coll. V, (1973), 336) of general formula (XIII) to yield thedesired amides (III) wherein R¹=H is readily achieved in an inertsolvent such as chloroform or dichloromethane in the presence of anorganic base such as triethylamine or ethyldiisopropylamine. In anidentical fashion amines of general formula (X) would react with acidchlorides (XIII) to yield the desired amides (III) wherein R¹=loweralkyl:

Alternatively the corresponding acids of general formula (XIV) may becoupled with dicyclohexylcarbodiimide (DCC)/hydroxybenzotriazole (HOBT)(see W. Konig et al, Chem. Ber., (1970), 103, 788) orhydroxybenzotriazole(HOBT)/2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) (see M. Bernatowicz et al., TetrahedronLett., (1989), 30, 4645) as condensing agents in dimethylformamide orchloroform to amines (IV) and (X) respectively yielding productsidentical to those described in the previous scheme:

The acids of general formula (XIV) are readily converted to thecorresponding acid chlorides (XIII) by the action of thionyl chloride oroxalyl chloride in the presence of catalytic dimethylformamide:

A third aspect of the present invention provides a compound of formula(A) or a solvate or physiologically functional derivative thereof foruse as a therapeutic agent, specifically as a modifier of the activityof the serotonin 5-HT_(2A) receptor. Modifiers of the activity of theserotonin 5-HT_(2A) receptor are believed to be of potential use for thetreatment or prophylaxis of CNS, gastrointestinal, cardiovascular, andinflammatory disorders. Compounds of the formula (A) may be administeredby oral, sublingual, parenteral, rectal, or topical administration. Inaddition to the neutral forms of compounds of formula (A) by appropriateaddition of an ionizable substituent, which does not alter the receptorspecificity of the compound, physiologically acceptable salts of thecompounds may also be formed and used as therapeutic agents. Differentamounts of the compounds of formula (A) will be required to achieve thedesired biological effect. The amount will depend on factors such as thespecific compound, the use for which it is intended, the means ofadministration, and the condition of the treated individual. A typicaldose may be expected to fall in the range of 0.001 to 200 mg perkilogram of body weight of the treated individual. Unit does may containfrom 1 to 200 mg of the compounds of formula (A) and may be administeredone or more times a day, individually or in multiples. In the case ofthe salt or solvate of a compound of formulas (A), the dose is based onthe cation (for salts) or the unsolvated compound.

A fourth aspect of the present invention provides pharmaceuticalcompositions, comprising at least one compound of formula (A) and/or anacceptable salt or solvate thereof (e.g., a pharmaceutically acceptablesalt or solvate) as an active ingredient combined with at least onecarrier or excipient (e.g., pharmaceutical carrier or excipient).Pharmaceutical compositions may be used in the treatment of clinicalconditions for which a modifier of the activity of the serotonin5-HT_(2A) receptor is indicated, particularly where the activeingredient is preferentially selective for the 5HT_(2A) receptor overthe 5HT_(2A) receptor, and most particularly where the active ingredientis also an inverse agonist at the 5HT_(2A) receptor. At least onecompound of formula (A) may be combined with the carrier in either solidor liquid form in a unit dose formulation. The pharmaceutical carriermust be compatible with the other ingredients in the composition andmust be tolerated by the individual recipient. Other physiologicallyactive ingredients may be incorporated into the pharmaceuticalcomposition of the invention if desired, and if such ingredients arecompatible with the other ingredients in the composition. Formulationsmay be prepared by any suitable method, typically by uniformly mixingthe active compound(s) with liquids or finely divided solid carriers, orboth, in the required proportions, and then, if necessary, forming theresulting mixture into a desired shape.

Conventional excipients, such as binding agents, fillers, acceptablewetting agents, tabletting lubricants, and disintegrants may be used intablets and capsules for oral administration. Liquid preparations fororal administration may be in the form of solutions, emulsions, aqueousor oily suspensions, and syrups. Alternatively, the oral preparationsmay be in the form of dry powder that can be reconstituted with water oranother suitable liquid vehicle before use. Additional additives such assuspending or emulsifying agents, non-aqueous vehicles (including edibleoils), preservatives, and flavorings and colorants may be added to theliquid preparations. Parenteral dosage forms may be prepared bydissolving the compound of the invention in a suitable liquid vehicleand filter sterilizing the solution before filling and sealing anappropriate vial or ampoule. These are just a few examples of the manyappropriate methods well known in the art for preparing dosage forms.

The fifth aspect of the present invention provides for the use of acompound of formula (A) in the preparation of a medicament for thetreatment of a medical condition for which a modifier of the activity ofthe serotonin 5-HT_(2A) receptor is indicated.

The sixth aspect of the present invention provides for a method oftreatment of a clinical condition of a mammal, such as a human, forwhich a modifier of the activity of the serotonin 5-HT_(2A) receptor isindicated, which comprises the administration to the mammal of atherapeutically effective amount of a compound of formula (A) or aphysiologically acceptable salt, solvate, or physiologically functionalderivative thereof.

Experimental Data

Mass spectra were recorded on a Micromass Platform™ LC with Gilson HPLC.Infra-red spectra were recorded on a Nicolet Avatar™ 360 FT-IR. Meltingpoints were recorded on a Electrothermal IA9200™ apparatus and areuncorrected. Proton nuclear magnetic resonance spectra were recorded ona Bruker™ 300 MHz machine. Chemical shifts are given with respect totetramethylsilane. In the text the following abbreviations are used; s(singlet), d (doublet), t (triplet), m (multiplet) or combinationsthereof. Chemical shifts are quoted in parts per million (ppm) and withcoupling constants in Hertz.

Thin layer chromatography was carried out using aluminium backed silicaplates (250 μL; GF₂₅₄). HPLC was recorded either on a HP Chemstation™1100 HPLC using a Hichrom 3.5 C18 reverse phase column (50 mm×2.1 mmi.d.). Linear gradient elution over 5 minutes−95% water (+0.1% TFA)/5%acetonitrile (+0.05% TFA) down to 5% water/95% acetonitrile. Flow rate0.8 mL/min [Method A]; or on a Hichrom 3.5 C18 reverse phase column (100mm×3.2 mm i.d.). Linear gradient elution over 11 minutes—95% water(+0.1% TFA)/5% acetonitrile (+0.05% TFA) down to 5% water/95%acetonitrile. Flow rate 1 mL/min [Method B]. Samples were routinelymonitored at 254 nM unless otherwise stated.

All reagents were purchased from commercial sources.

Experiment 1 Preparation and Analysis of 103487N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{(4-trifluoromethoxy)phenyl}amino]carboxamide

This compound is commercially available from Maybridge Chemical Company,Catalog No. KMO4515.

Experiment 2 Preparation and Analysis of 116100N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][4-methoxyphenoxy]carboxamide

To 4-methoxyphenylchloroformate (19 mg, 0.10 mmol) in CH₂Cl₂ (0.5 mL)was added dropwise a solution of3-(3-aminophenyl)-4-bromo-1-methylpyrazole (25 mg, 0.10 mmol) andtriethylamine (14 μL, 0.10 mmol) in CH₂Cl₂ (0.5 mL). The mixture wasstirred for 16 h and concentrated. Chromatography on flash silica (40%EtOAc/hexane) gave the title compound as a colourless solid (21 mg,52%), m.p. 140.3-141.8° C. (EtOAc/hexane).

IR: v_(max)=1748, 1592, 1504, 1412, 1190, 835, 764, 676 cm.⁻¹ MS (ES+):m/z (%)=404 (M+H⁸¹Br, 100), 402 (M+H⁷⁹Br, 90).

¹H-NMR (CD₃ OD): δ=3.80 (3H, s, CH₃), 3.81 (3H, s, CH₃), 6.91-6.98 (2H,m, ArH), 7.07-7.18 (3H, m, ArH), 7.42-7.53 (4H, m, ArH). HPLC: retentiontime 3.28 mins [Method A].

Tlc: Rf 0.4 (EtOAc/hexane).

Experiment 3 Preparation and Analysis of 116101N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][4-trifluoromethoxyphenyl]carboxamide

To 4-(trifluoromethoxy)benzoyl chloride (19 μL, 0.12 mmol) in CH₂Cl₂ (1mL) was added dropwise a solution of3-(3-aminophenyl)₄-bromo-1-methylpyrazole (30 mg, 0.12 mmol) andtriethylamine (17 μL, 0.12 mmol) in CH₂Cl₂ (0.5 mL). The reactionmixture was stirred for 16 h and concentrated. Chromatography on flashsilica (50% EtOAc/hexane) gave the title compound as a colourless solid(40 mg, 76%), m.p. 138.6-139.6° C. (EtOAc/hexane).

MS (ES+): m/z (%)=442 (M+H⁸¹Br, 93), 440 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=3.79 (3H, s, CH₃), 7.27 (1H, m, ArH), 7.45-7.60 (3H,m, ArH), 7.65 (1H, s, ArH), 7.87 (2H, m, ArH), 8.09 (2H, m, ArH), 10.51(1H, s, NH).

HPLC: retention time 3.60 min [Method A]. TLC: Rf 0.40 (50%EtOAc/hexane).

Experiment 4 Preparation and Analysis of 116102N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][2-thienyl]carboxamide

To thiophene-2-carbonyl chloride (11 μL, 0.09 mmol) in CH₂Cl₂ (1 mL) wasadded dropwise a solution of 3-(3-aminophenyl)₄-bromo-1-methylpyrazole(25 mg, 0.09 mmol) and triethylamine (14 μL, 0.09 mmol) in CH₂Cl₂ (0.5mL). The reaction mixture was stirred for 16 h and concentrated.Chromatography on flash silica (50% EtOAc/hexane) gave the titlecompound as a colourless solid (24 mg, 68%), m.p. 127.8-128.6° C.(EtOAc/hexane).

MS (ES+): m/z (%)=364 (M+H ⁸¹Br, 96), 362 (M+H⁷⁹Br, 100).

¹H-NMR (CD₃ OD): δ=3.81 (3H, s, CH₃), 7.19 (2H, m, ArH), 7.48-7.58 (2H,m, ArH), 7.68-7.83 (3H, m, ArH), 7.93 (1H, dd, J=1.0, 3.8, ArH).

HPLC: retention time 3.12 min [Method A]. TLC: Rf 0.30 (30%EtOAc/hexane).

Experiment 5 Preparation and Analysis of 116115N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][{(4-trifluoromethoxy)phenyl)methyl}amino]carboxamide

To a stirred solution of triphosgene (12 mg, 0.04 mmol) in CH₂Cl₂ (0.5mL) was added dropwise a solution of3-(3-aminophenyl)-4-bromo-1-methylpyrazole (30 mg, 0.12 mmol) andtriethylamine (33 μL, 0.24 mmol) in CH₂Cl₂ (0.5 mL). After 1 h,4-(trifluoromethoxy)benzylamine (23 mg, 0.12 mmol) was added. Thereaction mixture was stirred for 16 h and concentrated. Chromatographyon flash silica (75% EtOAc/hexane) gave the title compound as acolourless solid (38 mg, 68%), m.p. 144.6-145.8° C. (EtOAc/hexane).

IR: v_(max)=1626, 1558, 1278, 1160, 969, 871, 789, 703 cm⁻¹ MS (ES+):m/z (%)=471 (M+H ⁸¹Br, 91), 469 (M+H⁷⁹Br, 100).

¹H-NMR (CD₃ OD): δ=3.81 (3H, s, CH₃), 4.42 (2H, s, CH₂), 7.06 (1H, d,J=7.1, ArH), 7.24 (2H, d, J=8.4, ArH), 7.37-7.52 (6H, m, ArH). HPLC:retention time 3.06 mins [Method A].

Tlc: Rf 0.5 (EtOAc).

Experiment 6 Preparation and Analysis of 116120N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][4-chlorophenyl]carboxamide

To 4-chlorobenzoyl chloride (15 mg, 0.08 mmol) in CH₂Cl₂ (1 mL) wasadded dropwise a solution of 3-(3-aminophenyl)₄-bromo-1-methylpyrazole(21 mg, 0.08 mmol) and triethylamine (12 μL, 0.08 mmol) in CH₂Cl₂ (0.5mL). The mixture was stirred for 16 h and concentrated. Chromatographyon flash silica (50% EtOAc/hexane) gave the title compound as acolourless solid (23 mg, 72%), m.p. 184.4-184.8° C. (EtOAc/hexane).

MS (ES+): m/z (%)=394 (M+H⁸¹Br ³⁷Cl, 34), 392 (M+H⁷⁹Br ³⁷Cl (81Br ³⁵Cl),100), 390 (M+H⁷⁹Br ³⁵Cl, 67).

¹H-NMR (DMSO d₆): δ=3.79 (3H, s, CH₃), 7.25 (1H, d, J=7.9, ArH),7.51-7.6 (3H, m, ArH), 7.69 (1H, s, ArH), 7.90 (2H, m, ArH), 8.00 (2H,m, ArH), 10.51 (1H, s, NH).

HPLC: retention time 3.40 min [Method A]. TLC: Rf 0.35 (50%EtOAc/hexane).

Experiment 7 Preparation and Analysis of 116137N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-2-[4-(trifluoromethoxy)phenyl]acetamide

A solution of 3-(3-aminophenyl)-4-bromo-1-methylpyrazole (35 mg, 0.14mmol) and triethylamine (23 μL, 0.17 mmol) in DMF (0.5 mL) was added inone portion to a stirred solution of 4-trifluoromethoxyphenylacetic acid(31 mg, 0.14 mmol), HBTU (53 mg, 0.14 mmol) and HOBT (19 mg, 0.14 mmol)in DMF (1 mL). The mixture was heated at 70° C. for 24 h and thenquenched with aqueous sodium bicarbonate solution. Ethyl acetate wasadded and the organic phase separated, washed with water (.times.3),brine, dried (MgSO₄) and evaporated. Chromatography on flash silica (50%EtOAc/hexane) gave the title compound as a colourless solid (43 mg,68%). m.p. 141.2-142.5° C. (EtOAc/hexane).

IR: v_(max)=1684, 1592, 1510, 1253, 1217, 1157, 987, 798, 700 cm⁻¹

MS (ES+): m/z (%)=456 (M+H⁸¹Br, 100), 454 (M+H⁷⁹Br, 94).

¹H-NMR (DMSO d₆): δ=3.72 (2H, s, CH₂), 3.75 (3H, s, CH₃), 7.17 (1H, d,J=7.7, ArH), 7.33 (2H, d, J=8.7, ArH), 7.38-7.51 (3H, m, ArH), 7.62-7.73(3H, m, ArH), 10.44 (1H, s, NH).

HPLC: retention time 3.52 min [Method A].

Experiment 8 Preparation and Analysis of 116174N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-2-(3-fluorophenyl)acetamide

A mixture of 3-(3-aminophenyl)₄-bromo-1-methylpyrazole (30 mg, 0.12mmol), 3-fluorophenylacetic acid (18 mg, 0.12 mmol),1-hydroxybenzotriazole hydrate (16 mg, 0.12 mmol) and2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (46 mg, 0.12 mmol) were dissolved in chloroform(1.5 ml). N,N-Diisopropylethylamine (0.02 ml, 0.13 mmol) was added andthe mixture stirred at room temperature for 16 h. The reaction mixturewas then poured into brine and the organic layer washed with furtherbrine, dried over magnesium sulphate and then concentrated in vacuo. Thecrude product was purified by column chromatography (ethylacetate-toluene, 1:1), giving the title compound (12 mg, 26%). Rf 0.41(ethyl acetate-toluene, 1:1).

HPLC (Method B): retention time 7.07 min (100%). ¹H-NMR(CDCl₃) δ=3.77(2H, s), 3.83 (3H, s), 7.02-7.20 (4H, m), 7.54 (1H, s), 7.60-7.63 (1H,m). MS (AP+): m/z (%)=390 (M+H ⁸¹Br 100), 388 (M+H⁷⁹Br, 100).

Experiment 9 Preparation and Analysis of 116175N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-2-(3-methoxyphenyl)acetamide

A solution of 3-methoxyphenylacetyl chloride (0.02 ml, 0.12 mmol) indichloromethane (0.75 ml) was added dropwise at 0° C. to a solution of3-(3-aminophenyl)-4-bromo-1-methylpyrazole (30 mg, 0.12 mmol) andtriethylamine (0.02 ml, 0.13 mmol) in dichloromethane (0.75 ml). Theresulting mixture was stirred at room temperature for 16 h and thenpoured into brine. The organic layer was washed with more brine thendried over magnesium sulphate and concentrated in vacuo. The crudeproduct was purified by column chromatography (ethyl acetate-toluene,1:1), giving the title compound (9 mg, 19%). Rf 0.30 (ethylacetate-toluene, 1:1).

HPLC (Method B): retention time 8.62 min (97.09%). ¹H-NMR(CDCl₃) δ=3.76(2H, s), 3.82 (3H, s), 3.85 (3H, s), 6.84-6.90 (3H, m), 7.07-7.44 (5H,m), 7.53 (1H, s), 7.60 (1H, br s). MS (AP+): m/z (%)=402 (M+H⁸¹Br, 100),400 (M+H⁷⁹Br, 95).

Experiment 10 Preparation and Analysis of 116176N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-2-(2-fluorophenyl)acetamide

A mixture of 3-(3-aminophenyl)-4-bromo-1-methylpyrazole (30 mg, 0.12mmol), 2-fluorophenylacetic acid (18 mg, 0.12 mmol),1-hydroxybenzotriazole hydrate (16 mg, 0.12 mmol) and2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (46 mg, 0.12 mmol) were dissolved in chloroform(1.5 ml). N,N-Diisopropylethylamine (0.02 ml, 0.13 mmol) was added andthe mixture stirred at room temperature for 16 h. The reaction mixturewas then poured into brine and the organic layer washed with furtherbrine, dried over magnesium sulphate and then concentrated in vacuo. Thecrude product was purified by column chromatography (ethylacetate-toluene, 1:1), giving the title compound (15 mg, 32%). R_(f)0.52 (ethyl acetate-toluene, 1:1).

HPLC (Method B): retention time 7.28 min (100%). ¹H-NMR(CDCl₃) δ=3.79(2H, s), 3.83 (3H, s), 7.11-7.23 (3H, m), 7.30-7.55 (6H, m), 7.61-7.64(1H, m). MS (AP+): m/z (%)=390 (M+H⁸¹Br, 100), 388 (M+H⁷⁹Br, 100).

Experiment 11 Preparation and Analysis of 116177N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-2-(4-nitrophenyl)acetamide

A mixture of 3-(3-aminophenyl)-4-bromo-1-methylpyrazole (30 mg, 0.12mmol), 4-nitrophenylacetic acid (22 mg, 0.12 mmol),1-hydroxybenzotriazole hydrate (16 mg, 0.12 mmol) and2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(46 mg, 0.12 mmol) were dissolved in chloroform (1.5 ml).N,N-Diisopropylethylamine (0.02 ml, 0.13 mmol) was added and the mixturestirred at room temperature for 16 h. The reaction mixture was thenpoured into brine and the organic layer washed with further brine, driedover magnesium sulphate and then concentrated in vacuo. The crudeproduct was purified by column chromatography (ethyl acetate-toluene,1:1), giving the title compound (9 mg, 18%). Rf 0.19 (ethylacetate-toluene, 1:1).

HPLC (Method B): retention time 7.22 min (94.30%). ¹H-NMR(CDCl₃) δ=3.83(3H, s), 3.87 (2H, s), 7.18-7.23 (1H, m), 7.42-7.65 (7H, m), 8.22-8.30(2H, m). MS (AP+): m/z (%)=417 (M+H⁸¹Br, 100), 415 (M+H⁷⁹Br, 100).

Experiment 12 Preparation and Analysis of 116178N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-2-(2-methoxyphenyl)acetamide

A mixture of 3-(3-aminophenyl)-4-bromo-1-methylpyrazole (30 mg, 0.12mmol), 2-methoxyphenylacetic acid (20 mg, 0.12 mmol),1-hydroxybenzotriazole hydrate (16 mg, 0.12 mmol) and2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(46 mg, 0.12 mmol) were dissolved in chloroform (1.5 ml).N,N-Diisopropylethylamine (0.02 ml, 0.13 mmol) was added and the mixturestirred at room temperature for 16 h. The reaction mixture was thenpoured into brine and the organic layer washed with further brine, driedover magnesium sulphate and then concentrated in vacuo. The crudeproduct was purified by column chromatography (chloroform-methanol,99:1), giving the title compound (18 mg, 38%) as a colourless solid. Rf0.65 (chloroform-methanol, 98:2).

HPLC (Method B): retention time 7.16 min (100%). δ_(H) (CDCl₃) 3.76 (2H,s), 3.83 (3H, s), 3.98 (3H, s), 6.97-7.06 (2H, m), 7.11-7.16 (1H, m),7.31-7.50 (4H, m), 7.53 (1H, s), 7.57-7.60 (1H, m), 7.91 (1H, br s). MS(AP−): m/z (%)=400 (M−H⁸¹Br, 90), 398 (M−H⁷⁹Br, 100).

Experiment 13 Preparation and Analysis of 116192{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(1,1-dimethylethoxy)carboxamide

To di-tert-butyl dicarbonate (36 mg, 0.17 mmol) in methanol (1 mL) wasadded dropwise a solution of 3-(3-aminophenyl)-4-bromo-1-methylpyrazole(42 mg, 0.17 mmol) in methanol (1 mL). The mixture was stirred for 16 hand concentrated. Chromatography on flash silica (40% EtOAc/hexane) gavethe title compound as a colourless solid (29 mg, 49%) (EtOAc/hexane).

MS (CI−): m/z (%)=352 (M−H⁸¹Br, 100), 350 (M−H⁷⁹Br, 96).

¹H-NMR (DMSO d₆): δ=1.46 (9H, s, 3.times. CH₃), 3.73 (3H, s, CH₃), 7.07(1H, m, ArH), 7.42 (1H, t, J=7.7, ArH), 7.53-7.60 (2H, m, ArH), 7.64(1H, s, ArH), 9.57 (1H, s, NH).

HPLC: retention time 7.15 min [Method B].

One or the other (as indicated) of the two following synthetic protocolswas used to generate each of the compounds below:

Protocol A:

To an isocyanate (1 mmol) in CH₂Cl₂ (4 mL) was added dropwise a solutionof 3-(3-aminophenyl)-4-bromo-1-methylpyrazole (1 mmol) in CH₂Cl₂ (4 mL).The mixture was stirred for 16 hours and concentrated. Chromatography onflash silica (20%-80% EtOAc/hexane) followed by recrystallisation gavethe pure urea.Protocol B:

To a stirred solution of triphosgene (0.33 mmol) in CH₂Cl₂ (4 mL) wasadded dropwise a solution of 3-(3-aminophenyl)-4-bromo-1-methylpyrazole(1 mmol) and triethylamine (2 mmol) in CH₂Cl₂ (4 mL). After 1 hour, ananiline was added (1 mmol). The reaction mixture was stirred for 16hours and concentrated. Chromatography on flash silica (20%-80%EtOAc/hexane) followed by recrystallisation gave the pure urea.

Experiment 14 Preparation and Analysis of 116079N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][(4-methylthiophenyl)amino]carboxamide

[Protocol A]—4-(methylthio)phenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=419 (M+H⁸¹Br, 100), 417 (M+H⁷⁹Br, 94).

¹H-NMR (MeOH d₄): δ=2.42 (3H, s, SCH₃), 3.81 (3H, s, NCH₃), 7.06 (1H, m,ArH), 7.22 (2H, m, ArH), 7.37 (2H, m, ArH), 7.42-7.61 (4H, m, ArH).

HPLC: retention time 3.35 min [Method A].

Experiment 15 Preparation and Analysis of 116081N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl][(4-chlorophenyl)amino]carboxamide

[Protocol A]—4-chlorophenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=409 (M+H⁸¹Br ³⁷Cl, 19), 407 (M+H⁷⁹Br ³⁷Cl (81Br ³⁵Cl),100), 405 (M+H⁷⁹Br ³⁵Cl, 81).

¹H-NMR (MeOH d₄): δ=3.81 (3H, s, CH₃), 7.07 (1H, m, ArH), 7.23 (2H, m,ArH), 7.36-7.60 (6H, m, ArH).

HPLC: retention time 3.42 min [Method A].

Experiment 16 Preparation and Analysis of 116082{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(4-fluorophenyl)carboxamide

[Protocol A]—4-fluorophenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=391 (M+H⁸¹Br, 96), 389 (M+H⁷⁹Br, 100).

¹H-NMR (MeOH d₄): δ=3.81 (3H, s, CH₃), 6.93-7.11 (3H, m, ArH), 7.37-7.61(6H, m, ArH).

HPLC: retention time 3.11 min.

Experiment 17 Preparation and Analysis of 116087{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-[2-(trifluoromethoxy)phenyl]carboxamide

[Protocol A]—2-(trifluoromethoxy)phenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=457 (M+H⁸¹Br, 100), 455 (M+H⁷⁹Br, 95).

¹H-NMR (DMSO d₆): δ=3.79 (3H, s, CH₃), 7.06-7.18 (2H, m, ArH), 7.38-7.49(2H, m, ArH), 7.51-7.62 (2H, m, ArH), 7.65 (1H, m, ArH), 7.71 (1H, s,ArH), 8.24 (1H, dd, J=1.1, 8.2, ArH), 8.56 (1H, s, NH), 9.49 (1H, s,NH).

HPLC: retention time 3.40 min.

Experiment 18 Preparation and Analysis of 116089{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(2-nitrophenyl)carboxamide

[Protocol A]—2-nitrophenyl Isocyanate

yellow solid (EtOAc/hexane)

MS (ES+): m/z (%)=418 (M+H⁸¹Br, 98), 416 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=¹H-NMR (DMSO d₆): □=3.79 (3H, s, NCH₃), 7.14 (1H, m,ArH), 7.24 (1H, m, ArH), 7.50 (1H, t, J=7.7, ArH), 7.60 (2H, m, ArH),7.67 (1H, s, ArH), 7.71 (1H, s, ArH), 8.10 (1H, m, ArH), 8.29 (1H, m,ArH), 9.65 (1H, s, NH), 10.09 (1H, s, NH).

HPLC: retention time 3.10 min [Method A].

Experiment 19 Preparation and Analysis of 116091{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(4-methoxyphenyl)carboxamide

[Protocol A]—4-methoxyphenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=403 (M+H⁸¹Br, 100), 401 (M+H⁷⁹Br, 96).

¹H-NMR (DMSO d₆): δ=3.71 (3H, s, OCH₃), 3.79 (3H, s, NCH₃), 6.87 (2H, d,J=8.9, ArH), 7.06 (1H, d, J=7.5, ArH), 7.39 (2H, d, J=8.9, ArH),7.45-7.61 (3H, m, ArH), 7.65 (1H, s, ArH), 8.52 (1H, s, NH), 8.84 (1H,s, NH).

HPLC: retention time 3.08 min.

Experiment 20 Preparation and Analysis of 116092{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(2-methylphenyl)carboxamide

[Protocol A]—o-tolyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=387 (M+H⁸¹Br, 94),385 (M+H⁷⁹Br, 100).

¹H-NMR (MeOH d₄): δ=2.29 (3H, s, CH₃), 3.81 (3H, s, NCH₃), 7.03 (1H, dt,J=1.1, 7.5, ArH), 7.09 (1H, dt, J=1.1, 7.5, ArH), 7.13-7.22 (2H, m,ArH), 7.45 (1H, t, J=7.9, ArH), 7.49-7.57 (2H, m, ArH), 7.60-7.68 (2H,m, ArH).

HPLC: retention time 2.96 min.

Experiment 21 Preparation and Analysis of 116097{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-[4-(trifluoromethyl)phenyl]carboxamide

[Protocol A]—4-(trifluoromethyl)phenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=441 (M+H⁸¹Br, 94), 439 (M+H⁷⁹Br, 100).

¹H-NMR (MeOH d₄): δ=3.82 (3H, s, CH₃), 7.04-7.16 (3H, m, ArH), 7.20-7.47(6H, m, ArH).

HPLC: retention time 3.56 min.

Experiment 22 Preparation and Analysis of 116105{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(3-chlorophenyl)carboxamide

[Protocol A]—3-chlorophenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=409 (M+H⁸¹Br ³⁷Cl, 26), 407 (M+H⁷⁹Br ³⁷Cl (81Br ³⁵Cl),100), 405 (M+H⁷⁹Br ³⁵Cl, 70).

¹H-NMR (MeOH d₄): δ=3.81 (3H, s, NCH₃), 7.04 (1H, m, ArH), 7.10 (1H, m,ArH), 7.28 (2H, m, ArH), 7.47 (1H, t, J=7.8, ArH), 7.55 (1H, m, ArH),7.63 (1H, m, ArH), 7.68 (1H, s, ArH), 7.73 (1H, m, ArH), 9.04 (2H, s,NH).

HPLC: retention time 3.20 min [Method A].

Experiment 23 Preparation and Analysis of 116108{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(2-chlorophenyl)carboxamide

[Protocol A]—2-chlorophenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=409 (M+H⁸¹Br ³⁷Cl, 24), 407 (M+H⁷⁹Br ³⁷Cl (81Br ³⁵Cl),100), 405 (M+H⁷⁹Br ³⁵Cl, 72).

¹H-NMR (MeOH d₄): δ=3.81 (3H, s, NCH₃), 7.03 (1H, m, ArH), 7.11 (1H, m,ArH), 7.28 (1H, m, ArH), 7.35-7.53 (3H, m, ArH), 7.55 (1H, s, ArH), 7.62(1H, m, ArH), 8.11 (1H, m, ArH).

HPLC: retention time 3.13 min.

Experiment 24 Preparation and Analysis of 116110{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-[4-(methylethyl)phenyl]carboxamide

[Protocol A]—4-isopropylphenyl isocyanate

colorless solid (THF/hexane)

MS (ES+): m/z (%)=415 (M+H⁸¹Br, 100), 413 (M+H⁷⁹Br, 92).

¹H-NMR (MeOH d₄): δ=1.23 (6H, d, J=6.8, 2xCH₃), 2.86 (1H, septet, J=6.8,CH), 3.82 (3H, s, NCH₃), 7.09 (1H, m, ArH), 7.16 (2H, d, J=7.6, ArH),7.31 (2H, d, J=7.6, ArH), 7.42-7.51 (2H, m, ArH), 7.54 (1H, s, ArH),7.59 (1H, m, ArH).

HPLC: retention time 3.66 min.

Experiment 25 Preparation and Analysis of 116111{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(3-methoxyphenyl)carboxamide

[Protocol A]—3-methoxyphenyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=403 (M+H⁸¹Br, 100), 401 (M+H⁷⁹Br, 96).

¹H-NMR (MeOH d₄): δ=3.73 (3H, s, OCH₃), 3.81 (3H, s, NCH₃), 6.59 (1H, m,ArH), 6.91 (1H, m, ArH), 7.08 (1H, m, ArH), 7.14 (2H, m, ArH), 7.39-7.61(4H, m, ArH).

HPLC: retention time 2.90 min.

Experiment 26 Preparation and Analysis of 116112{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(3-methylphenyl)carboxamide

[Protocol A]—m-tolyl isocyanate

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=387 (M+H⁸¹Br, 100), 385 (M+H⁷⁹Br, 96).

¹H-NMR (DMSO d₆): δ=2.26 (3H, s, CH₃), 3.76 (3H, s, NCH₃), 6.79 (1H, m,ArH), 7.06-7.22 (3H, m, ArH), 7.29 (1H, m, ArH), 7.43-7.62 (3H, m, ArH),7.68 (1H, s, ArH), 8.65 (1H, s, NH), 8.89 (1H, s, NH).

HPLC: retention time 3.05 min [Method A].

Experiment 27 Preparation and Analysis of 116113{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-methyl-N-[4-(trifluoromethoxy)phenyl]-carboxamide

[Protocol B]—N-methyl-4-(trifluoromethoxy)aniline

pale yellow solid (EtOAc/hexane)

MS (ES+): m/z (%)=471 (M+H⁸¹Br, 88), 469 (M+H⁷⁹Br, 100).

¹H-NMR (MeOH d₄): δ=3.35 (3H, s, NCH₃), 3.81 (3H, s, NCH₃), 7.09 (1H, m,ArH), 7.25-7.51 (8H, m, ArH).

HPLC: retention time 3.56 min [Method A].

Experiment 28 Preparation and Analysis of 116119N-[4-(tert-butyl)phenyl]{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}carboxamide

[Protocol B]—4-tert-butylaniline

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=429 (M+H⁸¹Br, 98), 427 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=1.27 (9H, s, 3xCH₃), 3.79 (3H, s, NCH₃), 7.07 (1H,d, J=7.5, ArH), 7.29 (2H, d, J=8.7, ArH), 7.37 (2H, d, J=8.7, ArH), 7.45(1H, t, J=7.5, ArH), 7.51-7.60 (2H, m, ArH), 7.66 (1H, s, ArH), 8.65(1H, s, NH), 8.83 (1H, s, NH).

HPLC: retention time 3.77 min.

Experiment 29 Preparation and Analysis of 116122N-[4-(dimethylamino)phenyl]{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}carboxamide

[Protocol B]—NN-dimethyl-p-phenylenediamine

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=416 (M+H ⁸¹Br, 96), 414 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=2.86 (6H, s, NCH₃), 3.80 (3H, s, NCH₃), 6.80 (2H, m,ArH), 7.09 (1H, d, J=7.7, ArH), 7.28 (2H, m, ArH), 7.42 (1H, t, J=7.8,ArH), 7.52 (1H, m, ArH), 7.59 (1H, s, ArH), 7.67 (1H, s, ArH), 8.45 (1H,s, NH), 8.75 (1H, s, NH).

HPLC: retention time 2.07 min [Method A].

Experiment 30 Preparation and Analysis of 116138N-[4-(dimethylamino)phenyl]{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}carboxamide

[Protocol B]—3,5-dichloro-4-methylphenylamine

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=457 (M+H, 35), 455 (M+H, 100), 453 (M+H, 65).

¹H-NMR (DMSO d₆): δ=2.32 (3H, s, CH₃), 3.79 (3H, s, NCH₃), 7.11 (1H, d,J=7.4, ArH), 7.46 (1H, t, J=7.8, ArH), 7.50-7.64 (4H, m, ArH), 7.68 (1H,s, ArH), 9.02 (1H, s, NH), 9.09 (1H, s, NH).

HPLC: retention time 3.66 min.

Experiment 31 Preparation and Analysis of 116139{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-[4-(trifluoromethylthio)phenyl]carboxamide

[Protocol B]—4-(trifluoromethylthio)aniline

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=473 (M+H ⁸¹Br, 100), 471 (M+H⁷⁹Br, 94).

¹H-NMR (DMSO d₆): δ=3.81 (3H, s, NCH₃), 7.11 (1H, d, J=7.5, ArH), 7.47(1H, t, J=7.9, ArH), 7.51-7.63 (6H, m, ArH), 7.66 (1H, s, ArH), 9.03(1H, s, NH), 9.16 (1H, s, NH).

HPLC: retention time 3.76 min.

Experiment 32 Preparation and Analysis of 116141{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(cyclohexyl)carboxamide

[Protocol B]—cyclohexylamine

colorless solid, m.p. 155.5-156.3° C. (EtOAc/hexane).

MS (ES+): m/z (%)=379 (M+H⁸¹Br, 93), 377 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=1.07-1.34 (5H, m, 5xCH), 1.52 (1H, m, CH), 1.63 (2H,m, 2xCH), 1.76 (2H, m, 2xCH), 3.48 (1H, m, NCH), 3.74 (3H, s, CH₃), 6.15(1H, d, J=7.8, ArH), 6.98 (1H, d, J=7.5, ArH), 7.32-7.43 (2H, m, ArH),7.51 (1H, m, NH), 7.62 (1H, s, ArH), 8.50 (1H, s, NH).

HPLC: retention time 3.16 min [Method A].

TLC: retention factor 0.35 (50% EtOAc/hexane).

Experiment 33 Preparation and Analysis of 116143{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(phenylmethyl)carboxamide

[Protocol B]—benzylamine

colorless solid, m.p. 144.5-146.2° C. (EtOAc/hexane).

IR: _(max)=1622, 1565, 1467, 1374, 1239, 973, 802, 752, 695 cm⁻¹.

MS (ES+): m/z (%)=387 (M+H⁸¹Br, 89), 385 (M+H⁷⁹Br, 100).

¹H-NMR (CD₃OD): δ=3.81 (3H, s, CH₃), 4.40 (2H, s, CH₂), 7.05 (1H, m,ArH), 7.19-7.51 (9H, m, ArH).

HPLC: retention time 3.06 min [Method A].a

Experiment 34 Preparation and Analysis of 116144{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(2-fluorophenyl)carboxamide

[Protocol A]—2-fluorophenyl isocyanate

colorless solid (DCM/hexane)

MS (ES+): m/z (%)=391 (M+H⁸¹Br, 100), 389 (M+H⁷⁹Br, 90).

¹H-NMR (MeOH d₄): δ=3.79 (3H, s, NCH₃), 7.00-7.11 (4H, m, ArH),7.40-7.56 (3H, m, ArH), 7.61 (1H, m, ArH), 8.09 (1H, m, ArH).

HPLC: retention time 3.01 min.

Experiment 35 Preparation and Analysis of 1161452-({[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-amino}carbonylamino)benzamide

[Protocol B]—2-aminobenzamide

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=399 (M+H—17 ⁸¹Br, 100), 397 (M+H—17 ⁷⁹Br, 94).

¹H-NMR (DMSO d₆): δ=3.79 (3H, s, NCH₃), 6.93-7.10 (2H, m, ArH), 7.45(2H, t, J=7.8, ArH), 7.59-7.72 (5H, m, ArH), 8.22 (2H, m), 9.92 (1H, s,NH), 10.69 (1H, s, NH).

HPLC: retention time 2.88 min.

Experiment 36 Preparation and Analysis of 116147{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(4-cyanophenyl)carboxamide

[Protocol B]—4-aminobenzonitrile

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=398 (M+H⁸¹Br, 100), 396 (M+H⁷⁹Br, 96).

¹H-NMR (MeOH d₄): δ=3.81 (3H, s, NCH₃), 7.12 (1H, m, ArH), 7.46-7.57(3H, m, ArH), 7.62-7.69 (5H, m, ArH).

HPLC: retention time 3.12 min.

Experiment 37 Preparation and Analysis of 116148{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(2-cyanophenyl)carboxamide

[Protocol B]—2-aminobenzonitrile

colorless solid (EtOAc/hexane)

MS (ES+): m/z (%)=398 (M+H⁸¹Br, 95), 396 (M+H⁷⁹Br, 100).

¹H-NMR (CDCl₃): δ=3.79 (3H, s, CH₃), 7.13-7.28 (2H, m, ArH), 7.49 (1H,t, J=7.8, ArH), 7.57 (1H, m, ArH), 7.62 (1H, m, ArH), 7.65-7.71 (2H, m,ArH), 7.78 (1H, m, ArH), 8.07 (1H, d, J=8.6, ArH), 8.83 (1H, s, NH),9.62 (1H, s, NH).

HPLC: retention time 3.05 min [Method A].

Experiment 38 Preparation and Analysis of 116182{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(4-fluorophenylmethyl)carboxamide

[Protocol B]—4-fluorobenzylamine

colorless solid, m.p. 185.5-186.6° C. (EtOAc/hexane).

MS (ES+): m/z (%)=405 (M+H⁸¹Br, 97), 403 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=3.75 (3H, s, CH₃), 4.28 (2H, d, J=6.0, CH₂), 6.73(1H, t, J=5.9, NH), 7.01 (1H, d, J=7.5, ArH), 7.10-7.18 (2H, m, ArH),7.27-7.41 (4H, m, ArH), 7.56 (1H, s, ArH), 7.62 (1H, s, ArH), 8.82 (1H,s, NH).

HPLC: retention time 3.10 min [Method A].

TLC: retention factor 0.25 (50% EtOAc/hexane).

Experiment 39 Preparation and Analysis of 116183{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(3,4-dimethoxyphenylmethyl)carboxamide

[Protocol B]—3,4-dimethoxybenzylamine

colorless solid, m.p. 174.9-175.5° C. (EtOAc/hexane).

MS (CI+): m/z (%)=447 (M+H⁸¹Br, 100), 445 (M+H⁷⁹Br, 92).

¹H-NMR (DMSO d₆): δ=3.71 (3H, s, CH₃), 3.73 (3H, s, CH₃), 3.76 (3H, s,CH₃), 4.22 (2H, d, J=5.8, CH₂), 6.62 (1H, t, J=5.7, NH), 6.80 (1H, m,ArH), 6.89 (2H, m, ArH), 6.98 (1H, m, ArH), 7.36-7.51 (3H, m, ArH), 7.63(1H, s, ArH), 8.76 (1H, s, NH).

HPLC: retention time 2.86 min [Method A].

TLC: retention factor 0.20 (50% EtOAc/hexane).

Experiment 40 Preparation and Analysis of 116184{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(3,4,5-trimethoxyphenylmethyl)carboxamide

[Protocol B]—3,4,5-trimethoxybenzylamine

colorless solid (EtOAc/hexane).

MS (CI+): m/z (%)=477 (M+H⁸¹Br, 100), 475 (M+H⁷⁹Br, 95).

¹H-NMR (DMSO d₆): δ=3.63 (3H, s, OCH₃), 3.75 (9H, s, 3xCH₃), 4.21 (1H,d, J=5.9, CH₂), 6.61 (2H, s, ArH), 6.65 (1H, t, J=5.9, NH), 6.99 (1H, m,ArH), 7.40 (1H, t, J=7.7, ArH), 7.45 (1H, m, ArH), 7.56 (1H, m, ArH),7.64 (1H, s, ArH), 8.77 (1H, s, NH).

HPLC: retention time 5.91 min [Method B].

TLC: retention factor 0.50 (50% EtOAc/hexane).

Experiment 41 Preparation and Analysis of 116185N-[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]-[{(2-methylphenyl)methyl}amino]carboxamide

[Protocol B]—2-methylbenzylamine

colorless solid (EtOAc/hexane).

MS (CI+): m/z (%)=401 (M+H⁸¹Br, 96), 399 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=2.28 (3H, s, CH₃), 3.76 (3H, s, NCH₃), 4.28 (1H, d,J=5.8, CH₂), 6.60 (1H, t, J=5.8, NH), 7.01 (1H, m, ArH), 7.15 (3H, m,ArH), 7.24 (1H, m, ArH), 7.38-7.50 (2H, m, ArH), 7.57 (1H, m, ArH), 7.65(1H, s, ArH), 8.77 (1H, s, NH).

HPLC: retention time 2.74 min [Method A].

TLC: retention factor 0.20 (50% EtOAc/hexane).

Experiment 42 Preparation and Analysis of 116189{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-(4-methoxyphenylmethyl)carboxamide

[Protocol B]—4-methoxybenzylamine

colorless solid (EtOAc/hexane).

MS (CI+): m/z (%)=417 (M+H⁸¹Br, 94), 415 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO d₆): δ=3.72 (3H, s, CH₃), 3.77 (3H, s, NCH₃), 4.22 (1H, d,J=5.9, CH₂), 6.62 (1H, t, J=5.9, NH), 6.90 (2H, d, J=8.8, ArH), 7.00(1H, m, ArH), 7.23 (2H, d, J=8.8, ArH), 7.39 (1H, t, J=7.8, ArH), 7.43(1H, m, ArH), 7.56 (1H, m, ArH), 7.64 (1H, s, ArH), 8.73 (1H, s, NH).

HPLC: retention time 6.41 min [Method B].

TLC: retention factor 0.25 (50% EtOAc/hexane).

Experiment 43 Preparation and Analysis of 116194{[3-(4-bromo-1-methylpyrazol-3-yl)phenyl]amino}-N-[2-(4-methoxy)phenylethyl]carboxamide

[Protocol B]—2-(4-methoxyphenyl)ethylamine

colorless solid (EtOAc/hexane).

MS (ES+): m/z (%)=431 (M+H⁸¹Br, 95), 429 (M+H⁷⁹Br, 100).

¹H-NMR (DMSO (16): δ=2.68 (2H, t, J=7.1, CH₂), 3.31 (2H, m, CH₂), 3.71(3H, s, CH₃), 3.77 (3H, s, CH₃), 6.16 (1H, t, J=5.8, NH), 6.87 (2H, d,J=8.6, ArH), 6.99 (1H, dt, J=1.4, 7.3, ArH), 7.16 (2H, d, J=8.6, ArH),7.33-7.48 (2H, m, ArH), 7.52 (1H, m, ArH), 7.63 (1H, s, ArH), 8.71 (1H,s, NH).

HPLC: retention time 6.62 min [Method B].

An important point that can be derived from the foregoing data is thatby using a constitutively activated form of the receptor in the directidentification of candidate compounds, the selectivity of the compoundsis exceptional: as those in the art appreciate, the homology between thehuman 5HT_(2A) and 5HT_(2C) receptors is about 95%, and even with suchhomology, certain of the directly identified compounds, e.g., 116081 and116082 evidence a 100-fold difference in selectivity preference (asmeasured by IC₅₀ values) for the 5HT_(2A) receptor compared with the5HT_(2C) receptor. This is important for pharmaceutical compositions inthat such selectivity can help to reduce side-effects associated withinteraction of a drug with a non-target receptor.

Different embodiments of the invention will consist of differentconstitutively activated receptors, different expression systems,different assays, and different compounds. Those skilled in the art willunderstand which receptors to use with which expression systems andassay methods. All are considered within the scope of the teaching ofthis invention. In addition, those skilled in the art will recognizethat various modifications, additions, substitutions, and variations tothe illustrative examples set forth herein can be made without departingfrom the spirit of the invention and are, therefore, considered withinthe scope of the invention.

1. An isolated polypeptide comprising the amino acid sequence encoded bySEQ ID NO:24.